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Routine Histology Services

Routine Histology Services

Routine histology services and special histology services play a critical role in tissue-based research or diagnosis. Staining is the core of both types of services, employing different methods. By coloring otherwise transparent tissue sections, staining allows pathologists and researchers to view tissue morphology (structure) or to look for the presence or prevalence of particular cell types, structures or even micro-organisms such as bacteria.

The term “routine staining” refers to the hematoxylin and eosin stain (H&E) that is used “routinely” with all tissue specimens to reveal the underlying tissue structures and conditions. The term “special stains” is used to refer to alternative staining techniques outside of H&E routine staining to provide specific information researchers or pathologists need.

Routine histology services cover the procedures from wet tissues to formalin fixed paraffin embedded (FFPE) sections and histochemical staining (H&E)  staining. These services generally include:

  1. Tissue trimming
  2. Tissue processing
  3. Tissue embedding in paraffin blocks 
  4. Sectioning your paraffin-embedded tissues
  5. Mounting sections on positively charged slides or any other treated slides of your choice
  6. Routine Hematoxylin/Eosin (H&E)  stain or other special staining.
  7. As per customer’s request, we can deliver unstained slides, stained slides or isolated DNA/RNA/Protein from FFPE samples or staining images.

Frequently Asked Questions (FAQ) 

FAQ 1. How do I prepare the tissue samples for tissue processing?

  1. The usual fixative for paraffin embedded tissues is neutral buffered formalin (NBF). This is equivalent to 4% fresh paraformaldehyde in a buffered solution
  2. Where the best possible morphology is required, animals should be anesthesized and subjected to cardiac perfusion with saline, followed by a 10% formalin flush. If biochemical studies need to be performed on the tissue, a 10% formalin flush should not be used as it may interfere with subsequent analysis.
  3. For routine stains where perfusion is not required, tissue is sectioned and drop-fixed in a 10% formalin solution. Fixative volume should be 20 times that of tissue on a weight per volume; use 2 ml of formalin per 100 mg of tissue.
  4. Due to the slow rate of diffusion of formalin (0.5 mm/hr), tissue should be sectioned into 3-5 mm slices before transferring into formalin. This will ensure the best possible preservation of tissue and offers rapid uniform penetration and fixation of tissue within 3 hours.Tissue should be fixed for a minimum 48 hours at room temperature.
  5. After 48 hours of fixation, move tissue into 70% ethanol for long term storage.

 FAQ 2. What is Hematoxylin and Eosin (H&E) stain?

Hematoxylin and Eosin (H&E) stain is the most commonly used staining system. It is an important part of VitroVivo routine histology services. H&E contains two dyes haemotoxylin and eosin. Eosin is an acidic dye: it is negatively charged (general formula for acidic dyes is: Na+dye-). It stains basic (or acidophilic) structures red or pink. This is also sometimes termed ‘eosinophilic’. Thus the cytoplasm is stained pink by H&E staining.

Hematoxylin can be considered as a basic dye (general formula for basic dyes is:dye+ Cl-). Hematoxylin is actually a dye called hematein (obtained from the log-wood tree) used in combination with aluminium ions (Al3+). It is used to stain acidic (or basophilic) structures a purplish blue. (Hematoxylin is not strictly a basic dye, but it is used with a ‘mordant’ that makes this stain act as a basic dye. The mordant (aluminium salts) binds to the tissue, and then hematoxylin binds to the mordant, forming a tissue-mordant-hematoxylin linkage.) Thus the nucleus is stained purple by H&E staining.

This means that the nucleus, and parts of the cytoplasm that contain RNA stain up in one color (purple), and the rest of the cytoplasm stains up a different color (pink)

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FFPE, Frozen and LCM Sample Analysis

Formalin-fixed paraffin-embedded (FFPE), Frozen and laser microdissection (LCM) Sample Analysis Services

As a histology and molecular histopathology company, VitroVivo not only provides full scale histology services, but also offer FFPE, Frozen and LCM sample analysis services. These services cover the downstream application of  molecular biology and next generation sequencing (NGS): 

  1. Extract or isolate DNA, RNA/miRNA and protein from FFPE tissues, frozen tissues and  LCM samples
  2. PCR and RT-PCR
  3. Next Generation Sequencing (NGS) services: 
  • RNA Sequencing
  • Single Cell RNA Sequencing
  • Human Exome Sequencing
  • MicroRNA Sequencing
  • Target Capture Sequencing
  • Whole Human Genome Sequencing
  • 16S Metagenomics
  • Customized NGS Data Analysis

Our experienced biomedical scientists are able to offer these high quality services to bio. It will greatly accelerate your research projects. For more information, please send your request to:  service@vitrovivo.com

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Histopathologic Interpretation and Description by Board Certified Pathologist

Histopathological Interpretation and Description by Board Certified Pathologists

  1. histopathological interpretation and description under microscopy
  2. General experimental pathology, reporting and description
  3. Review FISH, IHC/IF and histochemistry slides.
  4. Histo/pathological description for publication

Frequently Asked Questions (FAQ) 

FAQ 1. What kind of samples or material should I prepare for slide review?

VitroVivo acceptes wet tissue, frozen samples, FFPE blocks, OCT blocks, unstained slides, stained slides for histology and pathology review or  microscopic description. We also accept high quality whole slide microscope images. If necessary, we can perform the rest of the  experiment for histopathology review.

FAQ 2. Do you review animal slides or human tissue slides?

Our pathologists review both human and animal tissue slides.

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Specialized Services

Specialized Services

  1. Serial sectioning
  2. Step sectioning
  3. Embedding and sectioning for culture cells or 3D tissue cultures
  4. ​Decalfication for bone tissue
  5. RNAse cutting precaution
  6. Tissue sectioning for RNA, DNA or protein isolation or extraction

Frequently Asked Questions (FAQ) 

FAQ 1. What are serial sections and why are they important?

Any of several microscopic sections made and arranged in consecutive order are called serial section. A continuous series of sections reveal structures in three dimensions (3D).

FAQ 2. What is step sectioning?

Step sectioning means taking a section and putting it on a slide, then shaving into the tissue a set distance, perhaps 100 microns or 500 microns, before taking the next section, and repeating this process. The result is a series of slides that show sections in “steps” or increments through the tissue.

FAQ 3. How do I prepare bone samples for tissue processing?

  1. fixation ( see “Routine FFPE Histology Services”, FAQ 1)
  2. After fixation, you can perform decalcification or you can send to VitroVivo for decalcification
  3. Method of decalcificationa:

          a. Quick decalcification for H&E staing or histology special staining: Immerse tissue cassette in 11% formic acid with a stir bar overnight in a fume hood, Rinse in running water for 30- 60 minutes (the smell should be gone).
          b. Slow EDTA decalcification for Immumostaining:  Fixed specimens are rinsed in old EDTA decal solution before placing in Decal Bath. Solution must be stirred continuously. Decal time for mouse bone samples are as follows: Calvaria: 7 days; Arm or legs: 14 days.

FAQ 4. How do I make EDTA decalcification Solution?

Make Solution Recipe (4 Liter):  Distilled Water  3 L; Hydroxide, concentrated 280 ml; EDTA (FW 292.2) 400 gm
Dilute 280 ml of concentrated Ammonium Hydroxide in 3L of distilled water. Add 400gm of EDTA and stir till dissolved. Add more concentrated ammonium hydroxide until pH is 7.2. Add more water to make the final volume 4L.

FAQ 5. How do I prepare 2D culture cell samples for making FFPE blocks?

  1. The volume of the packed cell pellet ideally needs to be approximately 0.5 mL. This requires approximately four 75 cm2 sized flasks, or two 150 cm2 flasks of near-confluent cell culture. Less material will result in a size-limited preparation, but this may be sufficient of only a few procedures are expected to be run.
  2. Scrape the cells: For adherent monolayer cells, do not trypsinize, as this may destroy cell-surface protein markers. Working quickly, pull the flasks from the incubator, and scrape the cells into the media. Transfer to a sterile 50 mL polypropylene centrifuge tube. Spin at room temperature for five minutes in swinging bucket centrifuge (setting 3 for 5 minutes in a standard clinical centrifuge, or approximately 200 x g).
  3. If using suspension cells, pellet 50 ml of cell culture supernatant in 50 ml conical tubes at 500xg for 10 min.
  4. Fixation: Aspirate media off cell pellet. Very slowly, add 20 ml neutral buffered formalin or zinc formalin (4º C) (contact the VitroVivo Biotech if you require this reagent), letting it flow gently down the side of the tube, in order not to disturb the pellet. You may re-centrifuge if the cell pellet is disturbed. The cells need to fix overnight at 4º C in formalin. You may bring the cell preparation the day of preparation, or after overnight incubation.
  5. If delivery of the preparation cannot be made within 24 hours of the start of fixation, remove the formalin and replace with 20 ml of 70% EtOH WITHOUT re-suspending the pellet. This will act as a non-crosslinking preservative, and the cells can be kept this way indefinitely at 4 º C. Do NOT freeze the cells.

FAQ 6. How do I prepare 3D culture cell samples for making FFPE blocks?

  1. If the 3D culture cells are suspension sphere cells, the cell preparation is same as 2D culture cell (see  FAQ3).
  2. If the 3 D culture cells grow on membrane ( such as PET membrane), cells should be fixed with 10% neutral buffered formalin for 30 min, then remove the formalin and replace with 70% EtOH.
  3. We will cut the membrane into 2-4 pieces and embed them in agarose gel for routine tissue processing.
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Molecular Histopathology Services

VitroVivo Molecular Histopathology Services

Molecular histopathology services provides a broad array of state-of-the-art technical and professional support to biomedical research and investigations. We offer the following services to meet your histology and pathology needs:

  1. ​Immunohistochemistry (IHC)
  2. Immunofluorescent (IF) stain (single or double antibody staining)
  3. Fluorescence in situ hybridization (FISH) 
  4. mRNA, lncRNA or microRNA In Situ  hybridization 
  5. In Situ apoptosis (TUNEL) assay
  6. In Situ BrdU or EdU cell proliferation assay
  7. In Situ autophagy detection assay​​

Frequently Asked Questions (FAQ)

FAQ 1 How do you design molecular histopathology experiment?

All of the molecular histopathology experiment will include positive control, negative control, and experiment samples. VitroVivo guarantees positive control works well to the intended standard.

FAQ 2. Should I send primary antibody to VitroVivo for IHC or IF services?

In general, we prefer that customers provide primary antibodies for IHC and IF services. VitroVivo will provide other reagents for IHC and IF experiments. If the primary antibodies have been validated by VitroVivo, we are happy to use our antibodies.

FAQ 3. Do I need to prepare probes for  in situ hybridization services?

Yes, you need to prepare probes. VitroVivo provides other reagents.

FAQ 4. What do I need to prepare for TUNEL, In Situ BrdU or EdU cell proliferation assay and  In Situ autophagy detection assay​​?

You only need to send the samples to us. 

FAQ 5, Does VitroVivo also provide human or animal samples?

Yes, We have customized sample collection services available.

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Special Staining Services

Images of Special Staining

VitroVivo Special Staining Services 

Special stains traditionally refer to any staining other than an H&E. It covers a wide variety of methods that may be used to visualize particular tissue structures, elements, or even micro-organisms not identified by H&E staining.

Other methods of staining such as immunohistochemistry or in situ hybridization used to target specific proteins or DNA/RNA sequences may be included as members of the “special stains”. However, since they are quite different in method and purpose, they could be separated into a third category know as “advanced stains”.

There can be hundreds of special stains for different purposes, but only a few are regularly used for research or clinical needs. Unlike H&E staining, special staining, due to their complexity, may have to be carried out by hands.

In contrast to “routine” stain services, special stains are used to differentiate specific components seen in the H&E-stained section. It is the application of various dyes to tissue sections prepared on a microscope slide. Special staining can be applied to highlight specific tissue components, microbes, metals, salts, and other artifacts.

VitroVivo offers the following special staining services with standard protocols:

  • Mammary gland whole mount staining
  • Alcian blue hematoxylin/orange G staining
  • Oil Red O staining
  • Modified Gomori’s trichrome staining
  • Bielschowsky’s silver staining
  • Masson’s trichrome staining
  • ​Picro-sirius red staining
  • Reticulum staining, and
  • More custom services as per specific request. Please send your email for inquiry to: service@vitrovivo.com
  • VitroVivo has special stain kits available for self staining

Frequently Asked Questions (FAQ)

FAQ 1. What is a special stain?

Any stain, other than an H&E stain, is classified as a special stain. The common special stains include mammary gland whole mount stain, alcian blue hematoxylin-orange G stain, alcian blue stain, alcian blue – PAS stain, oil red O stain, Nissl stain, Bielschowsky’s silver stain and Masson’s trichrome stain, etc. For more information, please visit our website page of Histochemical Stain Kits and Image Gallery.

FAQ 2. How do I choose VitroVivo special staining services?

This table gives you a guidance of the choice of special staining methods or kits (if you want to perform staining by your self). If you can not find the staining methods from this table, please send your email to  service@vitrovivo.com for inquiry, our service team will get back to you as soon as possible.

Product Name SKU# Visualization for Typical Results
Hematoxylin and Eosin Kit VB-3000 General morphology of tissue and cell
  • Sharp blue nucleus and red cytoplasm staining
Mammary Gland Whole Mount Stain Kit VB-3001 Wholemount staining of mouse mammary glands
  • Mouse mammary glands stain red
Alcian Blue Hematoxylin-Orange G Stain Kit VB-3002 Differentiate cartilage, mature bone, and immature bone found in various stages of endochondral ossification and fracture callus​
  1.  Bone:  orange to red
  2. Activated osteocytes:  bright blue pericellular ring
  3. Growth plate:  pale blue to blue
  4. Cartilage:  blue/purple (GAG/proteoglycan)
  5. Erythrocytes: bright pink
  6. Soft tissues (muscle, tendon, membranes):  pink to red
  7. Bone marrow:  dark blue
Alcian Blue Stain Kit VB-3003 Tisssue mucosubstances    
  1. Strongly acidic sulfated mucosubstances: blue
  2. Nuclei: pink to red
  3. Cytoplasm: pale pink
PAS Stain Kit VB-3004 Glycogen, mucin, and fungi
  1. Glycogen, mucin and some basement membranes: red/purple
  2. Fungi: red/purple
  3. Background: blue
Alcian Blue – PAS Stain Kit VB-3005 Acidic and neutral mucins as well as mixtures of acidic and neutral mucins
  1. Acidic mucins: blue
  2. Neutral mucins: magenta
  3. mixtures of above:blue/purple
  4. nuclei: deep blue
Luxol Fast Blue Stain Kit VB-3006 Myelin including phospholipids and neurons
  1. Myelin including phospholipids: blue to green
  2. Neuron: pink to violet
Oil Red O Stain Kit VB-3007 lipid and fat staining on formalin fixed frozen sections
  1. Lipid: red
  2. Nuclei:blue
Alizarin Red Stain Kit VB-3008 Calcium on tissue sections
  • Calcium deposits: orange-red
Prussian Blue Stain Kit VB-3009 Ferric iron on tissue sections
  1. Iron (hemosiderin): blue
  2. Nuclei: red
  3. Background: pink
Nissl Stain Kit VB-3010 Neuron Nissl body
  • Neuron (Nissl body):pink-violet
Congo Red Amyloid Stain Kit VB-3011 Amyloid deposits
  1. Amyloid, elastic fibers, eosinophil granules: red
  2. Nuclei: blue
Sudan Black B Lipid Stain Kit VB-3012 Lipid and fat
  1. Fat: blue or black
  2. Nuclei: red
Toluidine Blue Stain Kit VB-3013 Mast cells
  1. Mast cells: violet/red purple
  2. Background: blue
Modified Gomori’s Trichrome Stain Kit VB-3014 Connective fiber
  1. Nuclei: dark blue
  2. Muscle myofibrils: green-blue
  3. Mitochondria and endoplasmic reticulum stain: red
  4. Connective tissue stains: pale green-blue
  5. Myelin stains: purple red
  6. Type 1 fibers stain: darker blue/green as compared to type 2 fibers
Bielschowsky’s Silver Stain Kit VB-3015 Axons, neurofibrillary tangles and senile plaques
  1. Axons, neurofibrillary tangles and senile plaques: black
  2. Background: yellow to brown
Masson’s Trichrome Stain Kit VB-3016 Collagen and mucus
  1. Cytoplasm, keratin, muscle fibers, Erythrocytes:red
  2. Nuclei: black
  3. Collagen and mucus: blue
Picro-Sirius Red Stain Kit VB-3017 Collagen fibers
  1. Collagen: red
  2. Muscle Fibers: yellow
  3. Cytoplasm: yellow
Reticulum Stain Kit VB-3018 Reticular fibers
  1. Reticular fibers:black
  2. Nuclei: red
Verhoeff Van Gieson Elastin Stain Kit VB-3019 Elastic fibers
  1. Elastic fibers: blue-black to black
  2. Nuclei: blue to black
  3. Collagen: red
  4. Other tissue elements: yellow
Fontana-Masson Stain Kit VB-3020 Melanin pigment and argentaffin granules
  1. Melanin and argentaffin granules: Brown to black.
  2. Nuclei: Pink
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Cryotomy (Frozen Sectioning) Services

Cryotomy, Frozen Sectioning or Cryo-sectioning Services 

VitroVivo Biotech provides cryotomy, frozen sectioning or cryo-sectioning services.  Frozen sectioning is a method of choice or a lab procedure to perform rapid microscopic analysis of a specimen when paraffin processing may interfere with any downstream techniques or when some special tissue processing and outcome are required for particular research purposes. VitroVivo histology technicians take close attention to satisfy customers’ unique needs by optimizing their workflow—from embedding media and freezing spray to high performance of cryostats and accessories.

The frozen sectioning services usually take three steps:

 

Frequently Asked Questions (FAQ) 

FAQ 1. What is cryotomy?

Cryotomy, frozen sectioning or cryo-sectioning is a technique that a cryotome is used to prepare thin and frozen sections for biological tissues. Frozen sections can be used for tissue analysis that allows for rapid interpretation and diagnosis of the tissue during surgery. Cryotomy can also be used in the preparation of sections containing fats and enzymes which can easily be lost in alcohol or paraffin sections.

FAQ 2. How do I prepare OCT embedding block for unfixed fresh tissue?

Flash frozen fresh tissue in OCT is a common method for frozen section preparation. It features:

 Pros  Cons
  1. Fastest of all methods.
  2. Excellent for IHC, IF, ISH. No antigen retrieval required since there is no cross-linking fixative.
  3. Often easiest to section – depending upon the tissue.
  1. Poorest morphology.
  2. Prone to freezing artifact – must be snap frozen.
  3. ISH integrity – extreme clean techniques required or RNA will be rapidly and easily degraded.

Protocol

Place a drop of Optimal cutting temperature compound (OCT compound) in the bottom of the mold and place the tissue in the OCT. This will hold the tissue in place while you fill the mold with OCT. Just be careful to exclude large bubbles, fill the mold level full, and freeze by one of the methods below.

  • Method 1:

          Use dry ice in pellet form. Place a small stainless steel bowl (or Pyrex or polypropylene beaker) in the bottom of a styrofoam container and fill the space around the bowl with dry ice pellets. Place some pellets in the bowl and slowly add isopentane (2-methyl butane) or acetone. Work in a fume hood, of course, as these are flammable. When the pellets stop bubbling vigorously, the “slurry” is ready. Once you’ve filled the mold and oriented the tissue, immerse it in the liquid to freeze it.

  • Method 2.

          Isopentane also can be chilled in liquid nitrogen (-176ºC). With the liquid nitrogen in a styrofoam container or Dewar flask, use a tongs to lower a stainless steel, Pyrex, or polypropylene container of isopentane into the liquid nitrogen. The isopentane will start to become opaque as it nears freezing. Take the isopentane out of the liquid nitrogen and freeze the specimen as described above. Chill the isopentane again as necessary for subsequent tissues. This method has the advantage of very rapid freezing.

FAQ 3. How do I prepare OCT embedding block for fixed tissue?

Sometimes we need to fix the tissue first and then do the OCT embedding.

 Pros  Cons
  1.  Excellent morphology compared to other methods.
  2. May use a slower freeze in crushed powder dry ice alone, slush of dry ice and 100% alcohol, or in a beaker of isopentane surrounded by dry ice – without incurring freezing artifact or block cracking.
  3. Any of the freezing methods discussed can be used.
  4. Good for most IHC, IF and ISH.
  1.  Time consuming
  2. Most IHC will require antigen retrieval.
  3. Although the fixative cross-linking is protective for ISH techniques there is some RNA degradation

Protocol

Step 1

Fixation: Do all steps at 4°C
1. After removal of the tissues from the body, wash briefly in ice cold PBS plus Ca++ and Mg++
2. Fix tissues in fresh (<1wk old) 4% “paraformaldehyde” at 4°C or 10% neutral buffered formalin. The most ideal form of fixation for animal organs involves transcardiac perfusion of PFA prior to removal of the organ from the body.Time of subsequent immersion fixation depends on subsequent steps, but the best morphology is obtained if they are fixed 24 hrs after perfusion or 48-72 hrs if only immersion fixed.
3. Place tissues in 15% sucrose in PBS until tissue sinks (6-12 hrs) and then 30% sucrose in PBS for overnight or until tissue sinks. Best if the tissues are gently nutated, taking care to avoid contact with bubbles and the air surface interface.

Step 2 

OCT embedding: can use a slower freeze in crushed powder dry ice alone, or same method as preparation of OCT embedding block for unfixed fresh tissue (see FAQ 2 above).

FAQ 4. How can I store my OCT blocks?

The frozen blocks can be temporarily stored in dry ice. Transfer the blocks to a liquid nitrogen storage tank (Years) or -80°C freezer (Months).The sample should never be thaw unless there is specific requirement.

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Laser Capture Microdissetion (LCM) Services

VitroVivo ​Laser Capture Microdissection (LCM) Services

Laser Capture Microdissetion (LCM), also known as Laser Microdissection (LMD), is a contact- and contamination-free method for isolating specific single cells or entire areas of tissue from a wide variety of tissue samples. 
VitroVivo’s Laser Capture Microdissection (LCM/LMD) services provide customers with more power and precision by isolating specific cells from a mixed population. This enables more efficient and accurate downstream microgenomics applications such as next-generation sequencing, Sanger sequencing, PCR, proteomics and gene expression analysis. Moreover, LCM technique can isolate even a single cell from tissue, blood or semen samples on the basis of their morphology, genotype or immunohistochemical phenotype.

Laser Capture Microdissection (LCM/LMD) Associated Contract Research Projects

  1. Frozen and FFPE sample LCM/LMD
  2. Immunoguided LCM/LMD
  3. Live cell microdissection
  4. Plant cell LCM/LMD
  5. Downstream analysis of dissected samples

Laser Capture Microdissection (LCM/LMD) Approach and Advantages

LCM is a contact-free technique that helps minimize contamination and uncover answers that might otherwise be missed using a non-targeted sample due to the small size or homogenized nature of the sample. The laser cutting width is usually less than 1 µm, thus the target cells are not affected by the laser beam. Even live cells are not damaged by the laser cutting and are viable after cutting for cloning and reculturing as appropriate.

The laser capture microdissection process does not alter or damage the morphology and chemistry of the sample collected, nor the surrounding cells. For this reason, LCM is a useful method of collecting selected cells for DNA, RNA and/or protein analyses. LCM has also been used to isolate cellular structures, such as amyloid plaques. LCM can be performed on frozen and paraffin embedded archival tissues. A variety of tissue samples including blood smears, cytologic preparations, cell cultures and liquors of solid tissue may also be used.

Frequently Asked Questions (FAQ) 

FAQ 1. What is Laser Capture Microdissection (LCM) or Laser Microdissection (LMD) and its downstream applications ?

LCM or LMD is a method to isolate specific single cells or entire areas of tissue from a wide variety of tissue samples under direct microscopic visualization. LCM or LMD technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to generate histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling.

FAQ 2. How do I prepare frozen sections for Laser Capture Microdissection (LCM/LMD)?

  1. General Guidelines for Specimen Preparation: Tissue should be cut  and pieces frozen as soon as possible upon removal from the body or after death. RNase-Free conditions should be applied at all times during handling of tissues and sections.
  2. RNase-Free Technique: a). Wear disposable gloves and change frequently ; b). Use new or clean instruments between each animal or patient specimen; c). Use RNase-free or Nuclease free solutions, glassware and plasticware; d). Use RNase AWAY® or similar product to clean equipment
  3. Frozen Tissue Preparation: a). Tissues should be frozen in OCT or a similar product; b). Place a small amount of OCT on bottom of cryomold making sure there are no bubbles; c).Place tissue in the mold oriented such that the histologic region of interest is cut en face; d). Fill the mold with OCT to completely cover the mold making sure there are no bubbles (i.e. place OCT going from center to the periphery of the mold.
  4. Preferred Freezing Methods: a). Isopentane cooled over liquid Nitrogen; b). Isopentane cooled with dry ice; c). Other Freezing Methods: Dry Ice Alone (not optimal, slow) , Liquid Nitrogen (not optimal, sectioning difficulties) , Cryostat (NO!!!)
    Note: Tissues can be sectioned immediately or stored in a –70C freezer
  5. Frozen Tissue Sectioning: a).Gloves must be worn at all times; b).Cryostat must be cleaned prior to use. All surfaces must be wiped down with 95-100% ethanol, especially knife holder and anti-roll plate. c). Recommended Section thickness: LCM alone=8-10µm.  d). Mount sections onto slides at room temperature. After mounting the sections should be frozen as quickly as possible by placing directly on dry ice. SLIDES MUST REMAIN COLD!!! e). For mounting of sections onto frame membrane slides refer to Arcturus Protocol; f). Use separate areas of the microtome blade for each specimen; g). Sections can be stored in a slide box in a –70ºC freezer until further processing.
  6. General Staining Guidelines: a). Total staining time should be as short as possible; b). Staining dishes should be nuclease free; c). Staining solutions should be dedicated for use with LCM samples; d). Solutions are prepared with nuclease free water; e). Stained slides can be held in xylene until initiation of laser capture microdissection; f). Once removed from xylene, microdissection should be completed within 2 hours
  7. Histochemical Staining: a) 75% ETOH – 30 secs; b) NF dH20 – 30 secs; c) H&E stain -10-30 secs; d) NF dH20 – 30 secs; e) 75% ETOH – 30 secs; f) 95% ETOH – 30 secs; g) 100% ETOH – 30 secs; h) Xylene – 5 mins.

FAQ 3. How do I prepare FFPE sections for Laser Capture Microdissection (LCM/LMD)?

  1. FFPE Tissue Preparation
    Please Note: Formalin-fixation occurs by cross-linking proteins and nucleic acids with the aldehyde groups that not only affects the structural integrity of nucleic acids and proteins but the recovery as well. DNA can be analyzed most easily but there are greater challenges when analyzing RNA and proteins since the extraction process causes degradation of biomolecules.
    a) Tissue should be placed in 10% Neutral Buffered Formalin as soon as possible after harvesting
    b) Fixation should not exceed 24 hours at room temperature with tissue thickness not exceeding 5mm during fixation process
    c) Tissues should undergo tissue processing with embedding in paraffin immediately after fixation. Storage in ethanol or PBS is not recommended
  2. FFPE Tissue Sectioning: a) Use Nuclease Free or DEPC treated water for tissue floatation bath;  b)Float sections for minimal amount of time, no more than 1-2 mins; c) Once sections mounted on slides, prop up vertically to allow water to drain away from sections; d) Air dry for about 2 hrs at room temperature. e) Do not use oven to dry sections; g). Slides can be store for up to 2 wks at room temperature with dessicant, for longer terms store at –70ºC.
  3. General Staining Guidelines: a) Total staining time should be as short as possible; b) Staining dishes should be nuclease free; c) Staining solutions should be dedicated for use with LCM samples; d) Solutions are prepared with nuclease free water; e) Stained slides can be held in xylene until initiation of laser capture microdissection; f) Once removed from xylene, microdissection should be completed within 2 hours.
  4. Histochemical Staining: a) Fresh xylenes (to depariffinize the sections) – 5 min; b) Fresh xylenes – 5 min; c) 100% ethanol – 15 sec;  d) 95% ethanol – 15 sec; e) 70% ethanol – 15 sec; f) Deionized water – 15 sec; g) Mayer’s Hematoxylin – 30 sec; h) Deionized water – rinse (x 2) – 15 sec; i) 70% ethanol – 15 sec; j) Eosin Y – 5 sec; k) 95% ethanol – 15 sec; l) 95% ethanol – 15 sec; m) 100% ethanol – 15 sec; n) 100% ethanol – 15 sec; o) Xylenes (to ensure dehydration of the section) – 60 sec; p) Air-dry for approximately 2 minutes or gently use air gun to completely remove xylenes;

        The tissue is now ready for LCM process.

FAQ 4 Can you tell me the protocol for the preparation of rapid immunofluorence staining for direct laser capture of immunoreactive cells ?

  1. Outline tissue with a hydrophobic pen and allow to dry.
  2. Fix tissue in acetone-methanol (1:1) solution at -20 °C for 10 min. NOTE: Our experience has shown that the acetone-methanol fixation resulted in much more consistent immunohistochemistry than acetone or methanol alone.
  3. Rinse slide in phosphate buffered saline (PBS) with 1% Triton (RNase free). 
  4. Cover sections with 100-200 µl PBS with 1% Triton with 1º antibody diluted optimized dilution with 400 U/ml RNasin. Incubate for 5-10 min.
  5.  Rinse briefly in PBS twice and PBS-1% Triton.
  6. Cover tissue with 100-200 µl of goat anti-primary IgG labeled with Alexa Fluor 488 diluted 1:100 in PBS-1% Triton with 400 U/ml RNasin and 50 ng/ml DAPI. Incubate for 5 min.
  7. Rinse 2 times in PBS then dehydrate 30 s in a graded series of RNase free ethanol (75%-75%-95%-95%-100%-100%).
  8. Incubate in two washes of Xylene for 1 min then 5 min.
  9. Remove slides from Xylene immediately prior to use for LCM and allow to air dry.

FAQ 5. Does VitroVivo provide downstream application services of laser capture microdissection?

       Yes, please send your request to: service@vitrovuvo.com. You also can visit the page of FFPE, frozen and laser microdissection sample analysis services.

FAQ 6. Can you show me some publications related laser capture microdissection in past 2 years?

       Yes, see the links below:

  1. Genome-wide analysis revealed that DZNep reduces tubulointerstitial fibrosis via down-regulation of pro-fibrotic genes. Mimura I, et al.I.Sci Rep. 2018 Feb 28;8(1):3779.
  2. Alternative transcription of a shorter, non-anti-angiogenic thrombospondin-2 variant in cancer-associated blood vessels. Roudnicky F, et al. Oncogene. 2018 Feb 22.
  3. CrosstalkNet: A visualization tool for differential co-expression networks and communities. Manem VS, et al. Cancer Res. 2018 Feb 19.  
  4. High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening. Shinozaki Y, et al. Nat Commun. 2018 Jan 25;9(1):364.
  5. Lack of Fgf18 causes abnormal clustering of motor nerve terminals at the neuromuscular junction with reduced acetylcholine receptor clusters. Ito K, et al. Sci Rep. 2018 Jan 11;8(1):434
  6. Genomics-Driven Precision Medicine for Advanced Pancreatic Cancer: Early Results from the COMPASS Trial. Aung KL, et al. Clin Cancer Res. 2017 Dec 29.
  7. Morphological changes in different populations of bladder afferent neurons detected by herpes simplex virus (HSV) vectors with cell-type-specific promoters in mice with spinal cord injury. Shimizu N, et al. Neuroscience. 2017 Nov 19;364:190-201.
  8. Macrophage Infiltration Is a Causative Factor for Ligamentum Flavum Hypertrophy through the Activation of Collagen Production in Fibroblasts. Saito T, et al. Am J Pathol. 2017 Dec;187(12):2831-2840
  9. Defective decidualization during and after severe preeclampsia reveals a possible maternal contribution to the etiology.Garrido-Gomez T, et al. Proc Natl Acad Sci U S A. 2017 Oct 3;114(40):E8468-E8477.
  10. Cell-specific expression of plant nutrient transporter genes in orchid mycorrhizae. Fochi V, et al. Plant Sci. 2017 Oct;263:39-45. doi: 10.1016/j.plantsci.2017.06.015. Epub 2017 Jul 11.
  11. Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries. Boeva V, et al. Nat Genet. 2017 Sep;49(9):1408-1413.
  12. cGAS surveillance of micronuclei links genome instability to innate immunity. Mackenzie KJ, et al. Nature. 2017 Aug 24;548(7668):461-465. doi: 10.1038/nature23449. Epub 2017 Jul 24.
  13. The integrated pathway of TGFβ/Snail with TNFα/NFκB may facilitate the tumor-stroma interaction in the EMT process and colorectal cancer prognosis. Li H, et al.Sci Rep. 2017 Jul 7;7(1):4915.
  14. Interaction of reactive astrocytes with type I collagen induces astrocytic scar formation through the integrin-N-cadherin pathway after spinal cord injury. Hara M, et al. Nat Med. 2017 Jul;23(7):818-828.
  15. Increased T-cell Infiltration Elicited by Erk5 Deletion in a Pten-Deficient Mouse Model of Prostate Carcinogenesis. Loveridge CJ, et al. Cancer Res. 2017 Jun 15;77(12):3158-3168.
  16. Human Alternative Macrophages Populate Calcified Areas of Atherosclerotic Lesions and Display Impaired RANKL-Induced Osteoclastic Bone Resorption Activity.
  17. Chinetti-Gbaguidi G, et al. Circ Res. 2017 Jun 23;121(1):19-30. 
  18. Maternal smoke exposure decreases mesenchymal proliferation and modulates Rho-GTPase-dependent actin cytoskeletal signaling in fetal lungs. Unachukwu U, et al. FASEB J. 2017 Jun;31(6):2340-2351. 
  19. Synaptic Plasticity onto Dopamine Neurons Shapes Fear Learning. Pignatelli M,  et al. Neuron. 2017 Jan 18;93(2):425-440.
  20. Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling. Martin LB, et al.Nat Protoc. 2016 Dec;11(12):2376-2388.