Fixation: Fix the tissue sample in a suitable fixative (e.g., 10% neutral buffered formalin) for an appropriate period of time.
Dehydration: Transfer the fixed tissue to graded ethanol solutions (e.g., 70%, 80%, 95%, and 100% ethanol) to dehydrate it.
Clearing: Clear the tissue by transferring it to a clearing agent (e.g., xylene) to remove the ethanol.
2. Embedding
Infiltration: Transfer the cleared tissue into a suitable embedding medium (e.g., paraffin wax) and incubate it to allow infiltration.
Embedding: Place the infiltrated tissue in a mold containing molten paraffin wax and allow it to solidify.
3. Sectioning
Block Trimming: Trim the paraffin block to expose the tissue surface and ensure optimal sectioning.
Sectioning: Use a microtome to cut thin sections (usually around 4-6 μm) from the paraffin block. Collect the sections on glass slides coated with adhesive (e.g., gelatin).
4. Deparaffinization and Rehydration
Deparaffinization: Place the slides in a series of xylene or xylene substitute solutions to remove the paraffin from the tissue sections.
Rehydration: Rehydrate the tissue sections by passing them through a graded series of ethanol solutions (e.g., 100%, 95%, 80%, and 70% ethanol) and finally in distilled water.
5. Antigen Retrieval
Heat-Induced Antigen Retrieval (HIER): Perform antigen retrieval by immersing the slides in a suitable antigen retrieval buffer (e.g., citrate buffer) and heating them using a water bath or microwave to unmask epitopes. Allow the slides to cool down.
6. Blocking
Non-Specific Binding Block: Apply a blocking solution (e.g., 5-10% serum from the same species as the secondary antibody) to block non-specific binding sites on the tissue sections. Incubate the slides for a specified time.
7. Primary Antibody Incubation
Dilution: Dilute the primary antibody (specific to your target antigen) in a suitable antibody diluent (e.g., antibody dilution buffer, protein-based blocking solution) according to the manufacturer's instructions.
Incubation: Apply the diluted primary antibody to the tissue sections and incubate them in a humidified chamber at the recommended temperature for the specified duration.
8. Detection
Secondary Antibody: Wash the tissue sections with a suitable buffer (e.g., phosphate-buffered saline) and apply a secondary antibody conjugated to a detection label (e.g., enzyme, fluorescent dye) specific to the primary antibody's host species. Incubate the slides for the recommended time.
Amplification (if necessary): If signal amplification is required, apply an amplification reagent (e.g., avidin-biotin complex) according to the manufacturer's instructions.
Visualization: Use an appropriate detection method (e.g., enzyme substrate, fluorescent dye) to visualize the antigen-antibody complex.
9. Counterstaining
If desired, apply a suitable counterstain (e.g., hematoxylin) to visualize cellular structures or enhance contrast. Follow the staining protocol for the specific counterstain used.
10. Mounting
Dehydration: Dehydrate the slides by passing them through graded ethanol solutions (e.g., 70%, 95%, and 100% ethanol) to remove excess water.
Clearing: Clear the slides by placing them in a clearing agent (e.g., xylene) to remove the ethanol.
Mounting Medium: Apply a mounting medium (e.g., permanent mounting medium) to the tissue sections on the slides. Place a coverslip over the sections and gently press to remove any air bubbles.
11. Drying and Storage
Allow the slides to dry completely in a dust-free environment.
Store the slides in a slide box or slide cabinet at the appropriate temperature and conditions to maintain the stability of the staining.
Note: The protocol provided is a general guideline, and the specific steps and conditions may vary depending on the target antigen, antibodies, and staining system used. It is essential to refer to the manufacturer's instructions and optimize the protocol based on your specific experimental setup.
