Category: Services

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Tissue Microarray Services

Tissue Microarray (TMA) Services

Our comprehensive Tissue Microarray (TMA) services support both FFPE and frozen sample formats, enabling high-throughput tissue analysis for research and diagnostic applications. We provide precision construction, sectioning, staining, and expert pathology evaluation—delivering high-quality results tailored to advanced spatial biology platforms such as CosMx™ and Xenium™ and routine hight-troughput tissue analysis.

FFPE Sample TMA Services

  • FFPE Sample TMA Construction
    We construct custom TMAs from formalin-fixed paraffin-embedded (FFPE) tissue blocks with high precision, ensuring consistent core placement and layout.
  • FFPE Sample TMA Sectioning
    Our expert histotechnologists section FFPE TMAs with accuracy to preserve tissue morphology and antigenicity for downstream applications.
  • Restricted Area TMA Sectioning (CosMx/Xenium Compatible)
    We offer precise TMA sectioning targeting restricted regions of interest—fully compatible with high-resolution spatial platforms such as CosMx or Xenium.
  • FFPE TMA H&E Staining
    High-quality hematoxylin and eosin staining is performed on FFPE TMA slides for clear visualization of tissue architecture.
  • Pathology Description by Certified Pathologist
    Each TMA slide can be reviewed and described by a board-certified pathologist, providing detailed annotation and quality assessment.

Frozen Sample TMA Services

  • Frozen Sample TMA Construction
    We construct TMAs from fresh-frozen tissue blocks, maintaining tissue integrity and biomolecule preservation for specialized applications.
  • Frozen TMA Sectioning
    Our cryosectioning services deliver high-quality, uniform sections from frozen TMAs, ready for downstream analysis.
  • Frozen TMA H&E Staining
    We offer optimized H&E staining for frozen TMA sections, providing excellent contrast and tissue detail for histological evaluation.
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Routine Histology Services

Routine Histology Services – Expert Tissue Processing & Staining

Routine histology services are essential for tissue-based research and diagnostics, enabling researchers and pathologists to analyze tissue morphology, identify specific cell types, and detect microorganisms like bacteria. Histological staining enhances tissue visualization by adding contrast to otherwise transparent tissue sections.

Types of Histology Staining Services

1. Routine Staining (Hematoxylin & Eosin – H&E Stain)

  • The H&E stain is the standard technique used for all tissue specimens, revealing structural details and underlying tissue conditions.

2. Special Stains

  • Special stains provide additional insights beyond H&E, highlighting specific cellular components, pathogens, or tissue abnormalities.

Comprehensive Routine Histology Services

We offer a full range of histology lab services, from tissue processing to high-quality staining:
✅ Tissue Trimming – Precision trimming for optimal sectioning.
✅ Tissue Processing – Formalin fixation and paraffin embedding (FFPE).
✅ Paraffin Embedding – Long-term preservation of tissue samples.
✅ Tissue Sectioning – Thin section cutting from paraffin-embedded blocks.
✅ Slide Preparation – Mounting on positively charged or custom-treated slides.
✅ H&E & Special Staining – High-quality staining for enhanced tissue visualization.

🔬 Get Expert Routine Histology Services Today! Contact us for high-quality tissue processing, embedding, and staining solutions tailored to your research needs.

FFPE Histology Service Overview Video

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

FFPE, Frozen, and LCM Sample Analysis Services: DNA, RNA, Protein Extraction & More

At VitroVivo, a leading histology and molecular histopathology company, we offer comprehensive FFPE (Formalin-Fixed Paraffin-Embedded), Frozen, and Laser Capture Microdissection (LCM) sample analysis services. Our expert team of biomedical scientists provides high-quality services to accelerate your research, including:

  • DNA, RNA/miRNA, and Protein Extraction: Efficient isolation from FFPE tissues, frozen tissues, and LCM samples.
  • PCR and RT-PCR: Reliable amplification and analysis for gene expression and genetic studies.

Our services are designed to streamline your research projects and enhance the accuracy of your molecular studies. For more information or to request services, please contact us at service@vitrovivo.com.

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Histopathologic Interpretation and Description by Board Certified Pathologist

Histopathological Interpretation and Reporting by Board-Certified Pathologists

At VitroVivo, we provide expert histopathological interpretation and microscopic analysis by Board-Certified Pathologists. Our services include:

  • Detailed Histopathological Interpretation and Description: Expert analysis of tissue samples under the microscope.
  • General Experimental Pathology: Comprehensive reporting and description of pathology findings.
  • Slide Review for FISH, IHC/IF, and Histochemistry: In-depth examination and interpretation of molecular and histochemical assays.
  • Histo/Pathological Descriptions for Publication: High-quality, publication-ready descriptions tailored for research and academic purposes.

Our pathologists deliver precise and reliable histopathological interpretations to support your research, diagnostics, and publication needs. Contact us for professional and accurate pathological analysis services.

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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Histopathology Images and Analysis Services

Histopathology Imaging and Analysis Services: High-Quality Slide Scanning & Visual Scoring

At VitroVivo, we offer comprehensive Histopathology Image and Analysis Services, including:

  • High-Quality Whole Slide Scanning: Digital images of tissue samples for precise analysis.
  • Histology and Molecular Histopathology Analysis: Visual scoring for IHC (Immunohistochemistry), IF (Immunofluorescence), FISH/ISH (Fluorescence/In Situ Hybridization), and TUNEL assays.
  • Delineation of Tissue Regions of Interest: Accurate identification and analysis of key tissue areas for targeted research.

Our advanced services provide reliable and detailed histopathology data to support your biomedical research and diagnostic needs. Contact us today to learn more about our high-quality imaging and analysis solutions.

 
 
 
Olympus VS120 Whole Slide Scanning System
Picro-Sirius Red Stain, Rat Liver
Extraction of Picro-Sirius Red Stain

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Specialized Services

Specialized Histology Services – Precision Sectioning & Tissue Processing

VitroVivo Biotech offers specialized histology services designed to meet the advanced research needs of scientists and pathologists. Our expertise ensures high-quality tissue preparation, sectioning, and processing for applications such as molecular analysis, 3D tissue cultures, and bone research.

Our Specialized Histology Services

✔ Serial Sectioning – Consecutive tissue sections for detailed analysis.
✔ Step Sectioning – Sectioning at specific intervals for targeted examination.
✔ Embedding & Sectioning for Cultured Cells & 3D Tissue Cultures – High-precision processing for cell culture and organoid research.
✔ Bone Tissue Decalcification – Essential for preparing mineralized tissues for histological examination.
✔ RNase-Free Sectioning – Specialized precautions to prevent RNA degradation.
✔ Tissue Sectioning for Molecular Analysis – High-quality RNA, DNA, and protein isolation or extraction from tissue samples.

Why Choose VitroVivo’s Specialized Services?

✅ Expert Techniques – Optimized protocols for research precision.
✅ Molecular Compatibility – Ensuring high-quality tissue for genomic, proteomic, and transcriptomic studies.
✅ Custom Solutions – Tailored services for unique research requirements.

📩 Contact us today for high-quality specialized histology services to support your research!

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.

Flow Chat: From 3D Culture Cells to Digital Images

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Molecular Histopathology Services

VitroVivo Molecular Histopathology Services – Advanced Tissue Analysis for Biomedical Research

VitroVivo Biotech offers cutting-edge molecular histopathology services, providing comprehensive technical and professional support for biomedical research, pathology, and molecular investigations. Our specialized services enable precise protein, RNA, and DNA analysis within tissue samples, supporting cancer research, neuroscience, developmental biology, and more.

Our Molecular Histopathology Services

🔬 Immunohistochemistry (IHC) – High-sensitivity protein detection in tissue sections.
🧪 Immunofluorescence (IF) Staining – Single or double antibody staining for cellular and molecular imaging.
🧬 Fluorescence In Situ Hybridization (FISH) – DNA probe-based detection for gene amplification, deletion, or chromosomal abnormalities.
📌 In Situ RNA Hybridization (ISH) – Detection of mRNA, lncRNA, or microRNA, including RNAscope® ISH assays for high-resolution gene expression analysis.
💀 In Situ Apoptosis Detection (TUNEL Assay) – Identification of cell death in tissue samples.
🧩 In Situ BrdU or EdU Cell Proliferation Assay – Visualization of actively dividing cells.
🔄 In Situ Autophagy Detection Assay – Analysis of autophagy-related cellular processes.

Why Choose VitroVivo for Molecular Histopathology?

✅ State-of-the-Art Techniques – Advanced methodologies for high-quality, reproducible results.
✅ Customizable Solutions – Tailored services to meet specific research and diagnostic needs.
✅ Expert Support – Experienced histopathology specialists ensuring precision and reliability.

📩 Contact VitroVivo Biotech today to discuss your molecular histopathology service needs and receive expert support for your research!

Why Choose VitroVivo for RNAscope ISH Services? Watch the video below to learn more!

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.

IHC & ISH Images

CD3 IHC, Mouse Spleen

CD8 IHC, Mouse Spleen

FOXP3 IHC, Mouse Spleen

RNAscope, mRNA ISH, FFPE Tumor Samples

RNAscope, mRNA ISH, FFPE Tumor and Stroma Samples

RNAscope, mRNA ISH, FFPE Normal Breast Samples
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Special Staining Services

Images of Special Staining

VitroVivo Special Staining Services – Expert Tissue Staining for Research & Diagnostics

VitroVivo Biotech provides special staining services designed to enhance tissue visualization beyond standard Hematoxylin and Eosin (H&E) staining. Special stains help identify specific tissue structures, cellular components, microorganisms, metals, salts, and more that are not easily detected by routine staining.

What Are Special Stains?

🔬 Beyond H&E Staining – Special stains use advanced techniques to differentiate specific tissue components.
🧪 Precision & Customization – Essential for research, pathology, and clinical diagnostics.
🧬 Diverse Applications – Supports studies in histology, microbiology, and molecular biology.

Types of Special Stains

Special stains can be categorized into different groups based on their application:
✔ Histological Stains – Identify structural components within tissue.
✔ Microbial Stains – Detect bacteria, fungi, and other microorganisms.
✔ Histochemical Stains – Highlight specific biomolecules, metals, and deposits.
✔ Advanced Stains – Includes immunohistochemistry (IHC) and in situ hybridization (ISH) for protein and DNA/RNA analysis.

VitroVivo Special Staining Services

We offer a range of high-quality special stains using standardized protocols:

✅ Mammary Gland Whole Mount Staining
✅ Alcian Blue Hematoxylin/Orange G Staining
✅ Oil Red O Staining (for lipid detection)
✅ Modified Gomori’s Trichrome Staining (for muscle and connective tissues)
✅ Bielschowsky’s Silver Staining (for neural structures)
✅ Masson’s Trichrome Staining (for collagen fibers)
✅ Picro-Sirius Red Staining (for collagen analysis)
✅ Reticulum Staining (for reticular fibers)
✅ Custom Special Stains – Available upon request.

DIY Special Staining Kits

Prefer to perform staining in your own lab? VitroVivo offers special stain kits for self-use, ensuring high-quality and reproducible results.

📩 Contact us at service@vitrovivo.com for inquiries, pricing, or custom staining solutions tailored to your research needs!

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, and Cryo-Sectioning Services

VitroVivo Biotech specializes in cryotomy, frozen sectioning, and cryo-sectioning services, providing rapid and high-quality tissue processing for microscopic analysis. This method is ideal when paraffin embedding may interfere with downstream applications or when specialized tissue processing is required for specific research needs.

Our expert histology technicians ensure precision and efficiency by optimizing every step of the workflow—from embedding media and freezing techniques to high-performance cryostat sectioning and staining.

Why Choose Frozen Sectioning?

🔬 Rapid Tissue Analysis – Ideal for time-sensitive research.
❄️ Preserves Tissue Integrity – Avoids artifacts caused by paraffin processing.
🧬 Compatible with Special Techniques – Essential for immunostaining, enzymatic studies, and molecular research.

Frozen Sectioning Process

Our cryotomy services follow a three-step process for optimal results:

1️⃣ OCT Embedding – Tissues are embedded in Optimal Cutting Temperature (OCT) compound for precise sectioning.
2️⃣ Frozen Sectioning – High-precision cryostat sectioning ensures thin, uniform tissue slices.
3️⃣ Staining & Analysis – Choose from routine histological staining (H&E), special staining, and immunostaining for detailed tissue evaluation.

Custom Frozen Sectioning Services

We tailor our cryosectioning services to meet your specific research needs. Whether you need unstained frozen sections, H&E-stained slides, immunostaining, or customized tissue processing, we deliver high-quality results.

📩 Contact VitroVivo Biotech today for expert cryotomy and frozen sectioning services designed to support your research!

Cryotomy Frozen Sectioning Services – Video

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.