Immunology

scientificprotocols authored almost 4 years ago

Methods:

  1. Deparaffinize and rehydrate sections:
    • 3 x 3´ Xylene
    • 3 x 2´ 100% Ethanol
    • 1 x 2´ 95%, 80%, 70% Ethanol (each)
    • 1 x 5´ 1X PBS
  2. Antigen retrieval methods:

Sodium Citrate Antigen Retrieval:

  1. Place slides in a glass slide holder and fill in the rest of the rack with blank slides (10 total) to ensure even heating.
  2. Place rack in 600 ml of 10 mM Sodium Citrate (pH 6.0, 100 mM stock) in a glass 2L beaker. Mark a line at the top of the liquid on the beaker.
  3. Microwave for 20 minutes total, replacing evaporated water every 10 min.
  4. Cool slides for 20 minutes in the beaker.
  5. Wash 3 x 5´ in dd H2O, 1 x 5´ in 1X PBS.

Proteinase K Antigen Retrieval:

  1. Make a fresh solution of: 25 ul of 20 mg/ml Proteinase K
    • 2.5 ml of 1 M Tris-Cl, pH 8.0
    • 0.5 ml of 0.5 M EDTA, pH 8.0
    • to 50 mls with dd H2O
  2. Incubate slides in solution at 37°C for 5 min (do NOT pre-warm Prot K solution). A Coplin staining jar works well for this step.
  3. Wash 3 x 5´ with 1X PBS.

Urea Antigen Retrieval:

  1. Make a fresh solution of 1 M urea
  2. Place slides in a glass slide holder and fill in the rest of the rack with blank slides (10 total) to ensure even heating.
  3. Place rack in 600 ml of 1 M urea in a glass 2L beaker. Mark a line at the top of the liquid on the beaker.
  4. Microwave for 10, 20 or 30 minutes total, replacing evaporated water every 5-10 minutes.
  5. Cool slides for 30 minutes to 1 hour in the beaker.
  6. Wash 3 x 5´ in dd H2O, 1 x 5´ in 1X PBS.

Trypsin antigen retrieval protocol:

  1. Place slides or specimens into rack or other suitable container. Slides from paraffin-embedded samples should be dewaxed and rehydrated.
  2. Incubate the specimens for 10 minutes at room temp in PBS with occasional agitation to thoroughly hydrate the specimen.
  3. Pour off the PBS and incubate the specimen in a solution of 0.1% trypsin (tissue culture grade), 0.1% calcium chloride, 20 mM Tris (pH 7.6-8.0) solution for 2-20 minutes at room temperature depending upon the thickness and type of tissue. Optimization will be necessary. Typical time is 5 minutes.
  4. Stop the digestion by gently rinsing the specimen under the cold tap for 5 minutes, followed by incubation in TBS or PBS.

Microwave method: 1. Deparaffinize the section and rehydrate in PBS - Immerse the slides in plastic tray containing 100 mM Tris-HCL pH 10. - Adjust the power level on the microwave that the solution is just boiling. - Microwave for 5 minutes at the predetermined power level. - Check the level of buffer in the tray, fill up with buffer if necessary to cover the slides with buffer. - Repeat step 4 and 5 three more times. - Let the tray cool down at room temperature (takes 15-20 minutes, do not cool rapidly this may reduce the retrieval effect) - Wash the slides twice in PBS for 2 minutes.

Autoclave method:

  1. Deparaffinize the section and rehydrate in PBS
  2. Immerse the slides in a autoclavable tray, containing 100 mM Tris-HCL pH 10.
  3. Autoclave at 120 C for 10 minutes.
  4. Let the tray cool down at room temperature.
  5. Wash the slides twice in PBS for 2 minutes
  6. Block endogenous peroxidases:
    • Soak slides in 3% H2O2/PBS for 10-15 minutes at room temp.
    • Wash 3 x 5’ with 1X PBS.
  7. Shake and wipe off excess 1X PBS. Circle all sections with a PAP pen. Add 50-75 ul of blocking buffer to each section immediately, so that the sections don’t dry out. Don’t touch sections with tip.

Blocking buffers: 5% BSA/0.5% Tween-20 in 1X PBS

  • 3% BSA in 1X PBS
  • 3% BSA/0.1% Tween in 1X PBS
  • MOM (for mouse and rat monoclonal antibodies, use Molecular
  • Probes secondary antibodies with MOM basic kit)

Note: A lot of researchers would like to use 2.5-10% Normal Serum/0.5% Tween-20 in 1X PBS as a blocking buffer.

  1. Incubate 1 hour at room temperature or overnight at 4ºC in a humidified chamber. Do not let the slides touch each other.
  2. Dilute primary antibody in blocking buffer (dilutions vary depending on your antibody and expression of the antigen).
  3. Discard the blocking buffer, but don’t wash. Add 50-75 ul per section and incubate 1 hour at room temperature or overnight at 4ºC in a humidified chamber.
  4. Drain primary antibody off section. Wash slides 3 x 5´ in 1X PBS. (You may need to wash slides in 1X PBS + 0.1%-0.5% Tween-20 for some primary antibodies)
  5. Dilute biotinylated secondary antibody 1:150 to 1:750 in blocking buffer. Add 50-75 ul per section and incubate 45´-1 hour at room temperature in a humid chamber.
  6. Drain secondary antibody and wash slides 4 x 5’ in 1X PBS.
    • For using secondary antibodies that are peroxidase conjugated, skip to step 8.
  7. Add 1 drop of ABC (Ready to Use; Vector to each section and incubate samples for 30 minutes at room temperature. Wash 3 x 5 minutes in 1X PBS
  8. Make DAB according to Vector protocol in ddH2O. WEAR GLOVES.
    • 2.5 ml dd H2O
    • 1 drop buffer; mix
    • 2 drops DAB; mix
    • 1 drop H2O, mix.
  9. (If you want a gray-black stain, add 2 drops of the Nickel Solution; mix)
  10. Add immediately to slides and wait for color change (approximately 2-10 minutes).
  11. Drain slides and wash with dd H2O for 5 minutes.
  12. Dispose of DAB waste with bleach.
  13. Counterstain with hematoxylin:
    • Dip 8-12x (fast) Hematoxalin
    • Rinse deionized water
    • 1 x 5´ Tap water (to allow stain to develop)
    • Dip 8-12x (fast) Acid ethanol (to destain)
    • Rinse 2 x 1´ Tap water
    • Rinse 1 x 2´ Deionized water.
  14. Dehydrate and clear sections:
    • 3 x 5´ 95% ethanol
    • 3 x 5´ 100% ethanol (blot excess ethanol before going into xylene)
    • 3 x 5´ Xylene
  15. Coverslip slides using Permount (xylene based).
  16. Place a drop of Permount on the slide using a glass rod
  17. Angle the coverslip and let fall gently onto the slide.
  18. Allow the Permount to spread beneath the coverslip, covering all the tissue.
  19. Dry overnight in the hood. Be careful of not contaminating the sections with dusts.

Supplies:

  1. PAP pen Research Products International #195505
  2. Mouse on Mouse (MOM) Immunodetection Vector Laboratories
  3. Vectastain Elite ABC Reagent R.T.U. Vector Laboratories, PK-7100
  4. DAB Substrate Kit for Peroxidase Vector Laboratories, SK-4100
  5. Permount (Histological mounting medium) Fisher Scientific #SP15-100 (Refer to Rosen Lab’s Protocol

Note: All the conditions are different from lab. to lab, so you do need to optimize them to get the best pictures according to the samples and the IHC stuff at your own lab.

DOI

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