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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 Microdissection (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;
  5. 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.

Example LCM Images

Example LCM Video