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Immunohistochemistry(IHC) Protocol

Prepare formalin-fixed, paraffin-embedded tissue sections (Step 1-8):

1. Fix freshly dissected tissue (<3mm thick) with 10% neutral buffered Formalin (4% formaldehyde) for no less than 48 hours at room temperature. Inadequately fixation can make tissues dehydrated during tissue processing and become hard and brittle. 
2. Rinse the tissue with running tap water for 30min-40min to eliminate the formaldehyde.
3. Dehydrate the tissue through 70%, 80%, 95% alcohol, 5 min each, followed with 3 times of 100% alcohol, 5 min each.
4. Cleared the tissue in xylene for 2 times, 5 min each.
5. Immerse the tissue in paraffin for 3 times, 5 min each.
6. Embed the tissue in a paraffin block. The paraffin tissue block can be stored at room temperature for years.
7. Section the paraffin-embedded tissue block at 5-8 μm thickness on a microtome and float in a 40°C water bath containing distilled water.
8. Transfer the sections onto glass slides suitable for immunohistochemistry. Allow the slides to dry overnight and store slides at room temperature until ready for use.

Immunostain formalin-fixed, paraffin-embedded tissue sections (Step 9-29):

9. Deparaffinize slides in xylene for 2 times, 5 min each.
10. Transfer slides to 100% alcohol, for 2 times, 3 min each, and then transfer once through 95%, 70% and 50% alcohols respectively for 3 min each.
11. Block endogenous peroxidase activity by incubating sections in 3% H2O2 solution in methanol at room temperature for 10 min to block endogenous peroxidase activity.
12. Rinse with PBS for 2 times, 5 min each.
13. (Optional, recommended) Perform antigen retrieval to unmask the antigenic epitope. The most commonly used antigen retrieval is a citrate buffer method. Arrange the slides in a staining container. Pour 300 ml of 10 mM citrate buffer, pH 6.0 into the staining container and incubate it at 95-100°C for 10 min (optimal incubation time should be determined by user). Remove the staining container to room temperature and allow the slides to cool for 20 min.
14. Rinse slides with PBS for 2 times, 5 min each.
15. (Optional) Add 100 μl blocking buffer (e.g. 10% fetal bovine serum in PBS) onto the sections of the slides and incubate in a humidified chamber at room temperature for 1 h.
16. Drain off the blocking buffer from the slides.
17. Apply 100 μl appropriately diluted primary antibody (in antibody dilution buffer, e.g. 0.5% bovine serum albumin in PBS) to the sections on the slides and incubate in a humidified chamber at room temperature for 1 h.
18. Wash the slides with PBS for 2 times, 5 min each.
19. Apply 100 μl appropriately diluted biotinylated secondary antibody (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min.
20. Wash slides with PBS for 2 times, 5 min each.
21. Apply 100 μl appropriately diluted Sav-HRP conjugates (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min (keep protected from light).
22. Wash slides with PBS for 2 times, 5 min each.
23. Apply 100 μl DAB substrate solution (freshly made just before use: 0.05% DAB - 0.015% H2O2 in PBS) to the sections on the slides to reveal the color of antibody staining. Allow the color development for < 5 min until the desired color intensity is reached. (Caution: DAB is a suspect carcinogen. Handle with care. Wear gloves, lab coat and eye protection.)
24. Wash slides with PBS for 3 times, 2 min each.
25. (Optional) Counterstain slides by immersing sides in Hematoxylin for 1-2 min.
26. Rinse the slides in running tap water for 10 min.
27. Dehydrate the tissue slides through 4 times of alcohol (95%, 95%, 100% and 100%), 5 min each.
28. Clear the tissue slides in 3 times of xylene and coverslip using mounting solution. The mounted slides can be stored at room temperature permanently.
29. Observe the color of the antibody staining in the tissue sections under microscopy.

                                                                                                                                                                                                                                                                                                                                                                                                             http://www.immunohistochemistry.us


Immunohistochemistry(IHC) Troubleshooting


Weak or No Staining


  • Fixation of tissue was inadequate or improper:  Increase duration of postfixation or try different fixatives.
  • Fixation of tissue was too long:  Reduce the duration of postfixation. If the tissue has already been overfixed, optimized antigen retrieval procedure to unmask the epitope.
  • Deparaffinization was inadequate:  Deparaffinize sections longer and use fresh xylene or dewaxing solution.
  • Antibody was unsuitable for IHC procedures:  Check if it has been validated in IHC, and what type of IHC (FFPE or frozen tissue). Test the antibody in a native (non-denatured) WB to make sure it is not damaged.
  • Antibody was inactive due to improper storage:  Aliquot antibodies into smaller volumes and store in freezer (-20°C) and avoid repeated freeze and thaw cycles. Or store antibodies according to manufacturer's instructions.
  • Antibody concentration was inadequate:  Increase the concentration. Or run a serial dilution test to determine the optimal dilution that gives the best signal to noise ratio. Or incubate longer (e.g. overnight) at 4°C.
  • Antibody incubation time was inadequate: Increase incubation time (e.g. overnight) at 4°C.
  • Antibody was inactive Replace with a new batch of antibody.
  • Primary was incompatible with secondary antibody:  Use a secondary antibody that was raised against the species in which the primary was raised. For example, primary is raised in rabbit, use anti-rabbit secondary antibody).
  • Secondary antibody for immunofluorescence was not stored in the dark:  Always prevent the secondary antibody from exposure to light.
  • Substrate system was incompatible:  Replace with a different substrate system which is compatible with enzyme.
  • Mounting medium was Incompatible with enzyme substrate system:  Choose a correct mounting medium which is compatible with substrate system.
  • Reagents were applied in wrong order or steps omitted:  Check notes or procedure used.
  • PBS buffer was contaminated with bacteria that damage the phosphate groups on the protein of interest:  Add 0.01% azide in the PBS antibody storage buffer or use fresh sterile PBS.
  • Protein of interest was located in the nucleus and the antibody cannot penetrate the nucleus:  Add a strong permeabilizing agent like Triton X to the blocking buffer and antibody dilution buffer.
  • Protein of interest was not present in the tissue:  Run a positive control recommended by the supplier of the antibody
  • Protein of interest was not abundantly present in the tissue:  Use an amplification step to maximize the signal. For example, use a polymer conjugated secondary antibody, or biotin conjugated secondary antibody and a conjugated streptavidin.


Overstaining


  • Antibody concentration was too high:  Reduce concentration or perform a titration to determine the optimal dilution for antibody
  • Incubation time was too long:  Reduce incubation time
  • Incubation temperature was too high:  Reduce incubation temperature
  • Substrate incubation time was too long:  Reduce substrate incubation time
  • Sections dried out:  Avoid sections being dried out


High Background


  • Washing of sections was inadequate:  Wash at least 3 times between steps.
  • Endogenous enzyme such as peroxidase or alkaline phosphatase was active:  Block endogenous enzyme activity using 3% hydrogen peroxide (block peroxidase) in methanol or levamisole solution (block AP) prior to incubation of primary antibody.
  • Tissue contains endogenous biotin:  Block endogenous biotin activity using the avidin/biotin blocking reagent prior to incubation of primary antibody.
  • Non-specific binding of primary antibodies to tissue or antibody concentration was too high:  Titrate the antibody to the optimal concentration, dilute the antibody further and incubate at 4°C (a slow but targeted binding is best).
  • Non-specific binding of secondary antibody to tissue:  Increase the blocking incubation period and consider changing blocking agent. Recommend 10% normal serum of the species of the secondary antibody for 1 hr, or 1-5% BSA for 30 min for cells in culture. Or try a secondary antibody that has been pre-adsorbed against the lg of the species.
  • Diffusion of tissue antigen due to inadequate fixation:  Increase duration of postfixation.
  • Fixation procedures caused autofluorescence of immunofluorescence:  Formalin/PFA usually autofluorescence in the green spectrum, so try a fluorophore in the red range or a fluorophore in the infrared range if you have an infrared detection system.
  • Mouse antibody was used on mouse tissues:  Treat tissue with Mouse on Mouse blocking reagent prior to the primary antibody incubation
  • Sections dried out:  Avoid sections being dried out.
  • Incubation temperature may be too high:  Incubate sections or cells at 4°C.
  • Overstaining due to over amplification:  Reduce amplification incubation time and dilute the secondary antibody or amplification kit.
  • Too much substrate was applied:  Dilute substrate more and reduce substrate incubation time.
  • Chromogen reacted with the PBS present in the cells/tissue:   Use Tris buffer to wash sections prior to incubating with the substrate, then wash sections/cells in Tris buffer.
  • Permeabilization damaged the membrane and removed the membrane protein:  Use a less stringent detergent (e.g. Tween 20 instead of Triton X). Or simply remove permeabilizing agent from your buffers.