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Chromogenic IHC Staining of Frozen Tissue Sections

The following protocol has been developed and optimized by R&D Systems IHC/ICC laboratory for chromogenic IHC experiments using frozen tissue samples.

This protocol provides a basic guide for the fixation, cryostat sectioning, and staining of frozen tissue samples. Each investigator must determine the precise experimental conditions required to generate a strong and specific signal for each antigen of interest. If R&D Systems primary antibodies are employed, please refer to the product data sheets to obtain the recommended working dilutions. In this protocol, signal visualization is achieved using R&D Systems . For all other reagents, please follow the manufacturer’s instructions.

Please read protocol in its entirety before beginning.

Protocol for Fixing and Sectioning Frozen Tissues

Reagents Required

  • Formaldehyde Fixative Solution: 85 mM Na2HPO4, 75 mM KH2P04, 4% paraformaldehyde, and 14% (v/v) saturated picric acid, pH 6.9. (Picric acid is optional, enhances preservation of morphology in some tissues).
  • Sucrose Solution: 130 mM Na2HPO4, 30 mM KH2P04, 10% (w/v) sucrose, 0.01% sodium azide and 0.03% Bacitracin, pH 7.2
  • OCT Embedding Compound
  • Isopentane
  • Dry Ice

Procedure

This technique utilizes formaldehyde-based fixation before the tissue is frozen and sectioned using a cryostat.

  1. To preserve tissue morphology and retain the antigenicity of the target molecules, fix the tissue by vascular perfusion with 500 – 700 mL of Formaldehyde Fixative Solution.
  2. Perfuse the animal with 400 mL of Sucrose Solution. This step cryoprotects tissues frozen for cutting on a cryostat.
    Note: When it is not possible to fix by perfusion, dissected tissue may be fixed by immersion in a 10% formalin solution for 4-8 hours at room temperature. It is commonly accepted that the volume of fixative should be 50 times greater than the size of the immersed tissue. Avoid fixing the tissue for greater than 24 hours since tissue antigens may either be masked or destroyed.
    Note: R&D Systems scientists perfuse fix all rodent tissue with the exception of lung, spleen, and embryonic tissue, which are immersion fixed.
  3. Dissect the tissue, mount in OCT embedding compound, and freeze at -20 to -80 °C.
  4. Cut 5-15 µm thick tissue sections using a cryostat.
    Note: The suggested cryostat temperature is between -15 and -23 °C. The section will curl if the specimen is too cold. If it is too warm, it will stick to the knife.
  5. Thaw-mount the sections onto gelatin-coated histological slides. Slides are pre-coated with gelatin to enhance adhesion of the tissue. Please refer to the for instructions on how to prepare gelatin-coated slides.
  6. Dry the slides for 30 minutes on a slide warmer at 37 °C. Slides containing cryostat sections can be stored at -20 to -70 °C for up to 12 months.

Protocol for Cryopreservation of Tissues Prior to Fixation

This method utilizes frozen tissues that are fixed after snap-freezing and sectioning with a cryostat.

Procedure

  1. Immediately snap freeze fresh tissue in isopentane mixed with dry ice, and keep at -70 °C. Do not allow frozen tissue to thaw before cutting.
  2. Embed the tissue completely in OCT compound prior to cryostat sectioning.
  3. Cut cryostat sections at 5-10 µm and mount on gelatin-coated histological slides.
    Note: The suggested cryostat temperature is between -15 and -23 °C. The section will curl if the specimen is too cold. If it is too warm, it will stick to the knife.
  4. Air dry the sections for 30 minutes at room temperature to prevent sections from falling off the slides during antibody incubations.
    Note: Slides can be stored unfixed for up to one year at -80 °C. Frozen tissue samples saved for later analysis should be stored intact.
  5. Immediately add 50 µL of ice-cold Fixation Buffer to each tissue section upon removal from the freezer.
  6. Fix for 8 minutes at 2-8 °C or, optimally, at -20 °C for 20 minutes.

Protocol for Chromogenic Staining of Cryostat Sections

Reagents Required

  • Wash Buffer: 1X PBS (0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4)
  • Incubation Buffer: 1% bovine serum albumin, 1% normal donkey serum, 0.3% Triton¬Æ X-100, and 0.01% sodium azide in PBS
  • Primary Antibodies
  • : Kits include Biotinylated Secondary Antibodies, Serum Blocking Reagent, Peroxidase Blocking Reagent, Avidin Blocking Reagent, Biotin Blocking Reagent, HRP-Streptavidin Conjugate (HSS-HRP), and Chromogen Solution. Kits are available with chromogenic substrates 3,3‚Äô Diaminobenzidine (DAB, brown precipitate) or 3-amino-9-ethylcarbazole (AEC, red precipitate).
  • DAB Enhancer (Catalog # )
  • Hematoxylin
  • Aqueous Mounting Medium (Catalog # )
  • (if required; )

Materials

  • Gelatin-coated Slides ()
  • Coverslips

Procedure

This staining protocol has been developed and optimized for use with R&D Systems .

  1. When staining cryostat sections stored in a freezer, thaw the slides at room temperature for 10-20 minutes.
  2. Rehydrate the slides in wash buffer for 10 minutes. Drain the excess wash buffer.
    Note: Excessive fixation may result in the masking of an epitope and strong non-specific background signal that can obscure specific labeling. If necessary, an can be performed at this time. However, many antigen retrieval techniques are too harsh for cryostat cut tissue sections.
    Note: Endogenous peroxidase and biotin can react with secondary reagents and cause non-specific background staining. R&D Systems contain reagents to address these potential artifacts (applied in protocol steps 4-8).
  3. Surround the tissue with a hydrophobic barrier using a barrier pen.
  4. To quench endogenous peroxidase activity, incubate the sample with 1-3 drops peroxidase blocking reagent (3% H2O2 in water or methanol) for 5-15 minutes.
  5. Rinse the sample, and gently wash in wash buffer for 5 minutes.
  6. To reduce non-specific hydrophobic interactions between the primary antibodies and the tissue, block the section with 1-3 drops of serum blocking reagent for 15 minutes. Drain the slides, and wipe away any excess blocking reagent before proceeding to the next step. Do not rinse.
  7. To block binding to endogenous biotin, incubate the sample with 1-3 drops of avidin blocking reagent for 15 minutes. Rinse the sample with wash buffer, drain slides, and wipe away any excess wash buffer.
  8. To block subsequent binding to the avidin applied in step 6, incubate the sample with 1-3 drops of biotin blocking reagent for 15 minutes. Rinse with wash buffer, drain the slides, and wipe away any excess wash buffer.
  9. Incubate the sample with primary antibodies in Incubation Buffer. Follow manufacturer’s recommendations regarding working dilution for the primary antibody. For chromogenic IHC staining of frozen tissue sections using R&D Systems antibodies, it is recommended to incubate overnight at 2-8 °C. This incubation regime allows for optimal specific binding of antibodies to tissue targets and reduces non-specific background staining. These variables may need to be optimized for your system
    Note: are critical for the accurate interpretation of IHC/ICC results. All IHC/ICC experiments should include a negative control using the incubation buffer with no primary antibody to identify non-specific staining of the secondary reagents. Additional controls can be employed to support the specificity of staining generated by the primary antibody. These include absorption controls, isotype matched controls (for monoclonal primary antibodies), and tissue type controls.
  10. Rinse the sample with wash buffer. Wash 3 times with wash buffer for 5 minutes each, and drain the slides.
  11. Incubate the sample with 1-3 drops of biotinylated secondary antibodies for 30-60 minutes, adjusting the incubation time depending on the thickness of the section (approximately 30 minutes for 5-10 µm thick sections and 60 minutes for 10-20 µm thick sections).
  12. Rinse with wash buffer 3 times for 15 minutes each and drain the slides.
  13. Incubate the sample with 1-3 drops of High Sensitivity Streptavidin-HRP conjugate (HSS-HRP) for 30 minutes. This signal amplification technique is referred to as the labeled streptavidin-biotin (LSAB) method.
    Note: High Sensitivity Streptavidin is a chemical analog of Streptavidin that has little net positive charge at neutral or slightly alkaline pH and will interact only with biotin attached to secondary antibodies. HSS-HRP shows little or no non-specific binding to phospholipids, nucleic acids, and carbohydrate binding proteins.
  14. Rinse and wash 3 times in wash buffer for 2 minutes each.
  15. Calculate the working volume of DAB/AEC Chromogen Solution given that 100-200 µL is required to cover the entire tissue section on a slide. Add 1-5 drops of DAB/AEC Chromogen Solution to cover the entire tissue section, and incubate for 3-20 minutes. Monitor intensity of the tissue staining under a microscope. Colored precipitate will localize to the sites of antigen expression as the chromogenic substrate is converted by HRP enzyme into insoluble end product.
    Note: DAB and AEC are hazardous materials. Gloves and safety glasses should be worn and all steps performed inside a fume hood. Please refer to the MSDS for safe deactivation.
    Note: If required, DAB Enhancer (Catalog # ) can be used to intensify the DAB Chromogen solution.
  16. Rinse the sample with wash buffer 3 times for 10 minutes each.
  17. Rinse in deionized H2O and drain the slides.
  18. Stained tissue can be mounted either without nuclear counterstaining or counterstained with nuclear counterstain hematoxylin for better visualization of the tissue morphology.
    Note: Hematoxylin counterstain can obscure visualization of targets localized in cell nuclei.
  19. Cover stained tissue with a coverslip of an appropriate size, place slides vertically on a filter paper or towel to drain excess mounting medium and allow them to dry.
    Note: Unlike DAB, AEC is soluble in alcohols and xylene. Tissue sections subjected to an HRP-AEC protocol should be coverslipped using only aqueous mounting media.
  20. Visualize staining of tissue under a microscope using a bright-field illumination.
    Note: Initial IHC/ICC studies often require further optimization and/or additional steps.

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