IHC with frozen samples

Stage 1 - Freezing

To prepare tissue sections on slides, we must first freeze the samples before sectioning and fixation.

Materials required

• Samples
• Isopentane
• Dry ice
• Optimal Cutting Temperature (OCT) compound
• Tissue embedding molds and cassettes

Steps

Prepare a cold isopentane bath.

• Fill a large insulated container with dry ice.
• Partially fill a metal container with isopentane and place the container on dry ice.
• Wait for 5 mins, allowing the isopentane to be cooled by the dry ice.

Place fresh tissue in an embedding mold and fill it with OCT compound.

• Orient the tissue as desired

Freeze the tissue using the isopentane bath.

• Hold the tissue mold over the isopentane bath with forceps for 10 – 20 seconds until the tissue block turns opaque.

Store the frozen tissue at -80°C until ready for sectioning.

Stage 2 - Sectioning

Once the tissue is embedded, we can cut it into sections and mount it to microscope slides.

Materials required

• Frozen tissue samples
• Suitable slides
• Cryostat
• Cryostat blade

Steps

Bring frozen tissue samples to -20°C in a cryostat and allow them to equilibrate overnight.

Attach the frozen block to the sample holder and leave it to set.

Set up the cryostat blade by placing it in the holder, ensuring it is secure, and setting the clearance angle.

• Follow the manufacturer’s instructions for guidance on setting the clearance angle.

Trim the frozen tissue block to expose the tissue surface.

Proceed to cut sections at a thickness of 5 – 8 µm.

Collect the sections using a brush and place them on a slide ready for subsequent fixation steps.

Stage 3 - Fixation

Here we need to air dry the frozen samples and then fix them to preserve protein and tissue morphology before antibody incubation.

Materials required

• Tissue sections on suitable IHC slides
• Wash buffer (PBST)
• Fixative (10% NBF or 4% PFA, 100% acetone or methanol)

Steps

Dry frozen samples at room temperature for 15 mins.

Select a suitable fixative at room temperature.

Immerse the tissue slide in the fixative solution.

• Incubate samples for 15 mins at room temperature.

Wash the tissue slides three times with PBST.

Stage 4 - Blocking

Blocking steps are particularly important in IHC to prevent high background staining in images.

Materials required

• Your tissue sections

• Protein blocking solution (example: 2 - 10 % normal serum of same species as secondary antibody, or sera-free protein block ab64226)

• Biotin blocking solution (optional – for biotinylated antibodies – example: ab64212)

• Endogenous peroxidase blocking solution (optional for peroxidase-conjugated antibodies – example: ab64218)

• Wash buffer, PBST (PBS, 0.05% Triton X-100 or NP-40)

Steps

Wash the slides twice for 5 mins in PBST.

Perform endogenous avidin/biotin block (optional).

• Incubate slides for 10 mins in avidin blocking solution at room temperature.
• Wash slides once with PBST.
• Incubate slides for 10 mins in biotin-blocking solution at room temperature.
• Wash slides once with PBST.

Perform endogenous peroxidase block (optional).

• Incubate slides with 3% hydrogen peroxide for 10 mins at room temperature.
• Wash slides once with PBST.

Perform protein block.

• Incubate slides in protein blocking reagent for 30 – 60 mins at room temperature.

Wash slides three times for 5 mins with PBST.

Proceed to immunostaining.

Stage 5 - Antibody incubation

After performing the necessary blocking steps, we’re ready to stain our tissues with antibodies. We can stain tissues directly with conjugated primary antibodies or indirectly with conjugated secondary antibodies.

Multicolor IHC involves staining cells with two or more antibodies to reveal the distribution of two or more proteins of interest. Both the indirect and direct protocols given below can be adapted for multicolor IHC. We can either incubate cells with multiple antibody sets simultaneously, or incubate cells with each antibody set sequentially, performing blocking between each incubation.

Materials required

• Tissues that have undergone relevant blocking steps
• Antibody dilution buffer (PBS + 1% BSA)
• Wash buffer (PBST)
• Conjugated primary antibody

Steps

Determine the optimal antibody dilutions to use, then dilute the antibodies in PBS with 1% BSA.

Incubate the slides in the pre-diluted primary antibody.

• Incubate for 1 hr at room temperature, or overnight at 4°C.

Wash the slides three times with PBST.

Proceed to counterstaining, mounting and imaging.

Materials required

• Tissues that have undergone relevant blocking steps

• Dilution buffer (PBS, 1% BSA)

• Wash buffer (PBST)

• Primary antibody

• Conjugated secondary antibody (example: our conjugated secondaries)

• Hydrophobic barrier pen (example: ab2601)- optional

Steps

Dilute the primary and secondary antibodies in PBS with 1% BSA.

• Suggested dilutions will often be suggested on the antibody datasheet.

Incubate the samples with the pre-diluted primary antibody.

• Incubate for 1 – 2 hrs at room temperature, or overnight at 4°C.

Wash the slides three times with PBST.

Incubate the samples with pre-diluted secondary antibody.

• Incubate according to the manufacturer’s guidance.
• Usually, it’s recommended to incubate for 45-60 mins at room temperature.

Wash the slides three times with PBST.

Stage 6 - Detection

After incubation with antibodies, you're now ready to image your antibodies according to the procedures below.

Materials required

• Tissue slides stained with enzyme-conjugated antibody

• Chromogenic substrate (examples for HRP: DAB ab64238, AEC ab64252)

• Counterstain (optional, example: Mayer’s Hematoxylin ab220365)

• Mounting medium (organic, example ab104141, or aqueous ab64230)

• Sealant (optional for aqueous mounting media, example nail polish or Limonene ab104141)

• Coverslip

• Microscope

Steps

Immerse the slide in chromogenic substrate solution.

• Incubate until the desired color is observed.

Wash the slides with cold running water to remove excess stain.

Immerse the slide in counterstain solution (optional).

• Incubate according to the manufacturer’s guidance at room temperature or until the desired color is observed.

Wash the slides with cold running water to remove the excess stain and to blue the hematoxylin.

Dehydrate and clear the tissue before applying organic mounting media (optional).

• Perform the following exchange at room temperature, manually in a Coplin’s jar, or in an automated embedding system.

Add a few drops of mounting medium to the slid: let the slide stand at room temperature for 5 mins.

Carefully place a coverslip over the slide using forceps.

• If using an aqueous mounting medium, seal the coverslip with limonene or nail polish.

• If using an organic mounting medium, allow the medium to dry completely.

Image the slides using a microscope.

Materials required

• Tissue slides stained with fluorophore-conjugated antibody

• Fluorescent counterstain (optional, example: DAPI ab228549)

• Mounting medium suitable for fluorescent detection (example: ab104135)

• Sealant (example: nail polish or Limonene ab104141)

• Coverslip

• Microscope

• Deionized (DI) water

Steps

Immerse the slide in counterstain solution (optional).

• Incubate according to the manufacturer’s guidance at room temperature or until the desired color is observed.

Wash the slides with cold DI water to remove the excess stain.

Add a few drops of mounting medium to the slides.

• Let slide stand at room temperature for around 5 mins.

Carefully place a coverslip over the slide using forceps.

• If using an aqueous mounting medium, seal the coverslip with limonene or nail polish.

Image the slides using a microscope.