STEP BY STEP IMMUNOHISTOCHEMISTRY
Day 1.
1 ) Preparing the slides
- Deparafinize slides in Xylene x2 15-20 min ea.
Rehydrate
- 100% Ethanol, 2 min
- 100% Ethanol, 2 min
- 95% Ethanol, 2 min
- 95% Ethanol, 2 min
- 70% Ethanol, 2 min
- Milipore Water, 5 min
2) Antigen Unmasking
- 0.1% Trypsin (in 1x TBS) 5-20 min. at RT (200ml TBS 1X and .2 g Trypsin)
* (I usually do 15 min.)
3) Block Endogenous peroxidase activity
- Prepare 1% H2O2 in milipore water for 15 min. RT gentle shaking
4) Blocking/Primary Ab
- Pour off H2O2 and fill with TT buffer; wash in TT buffer 3x5min, RT, gentle shaking. Dry off buffer around sample (do NOT let sample dry or touch it with paper towel) and draw barrier around sample area with PAP pen. Keep slides in TT before and after drawing barrier
- Lay slides flat in humidified chamber (slide box w/wet paper towels) and add blocking buffer (in fridge). Shake gently for 20 min. RT
- Dilute primary Ab in blocking buffer
- Tip off blocking buffer and add 250-400ul of diluted antibody (or blocking buffer for neg control) incubate 15-20 min at RT then O/N at 4*C
Day 2
5) Secondary Ab
- Wash sections with TT buffer 4x5min. Do not cross contaminate the samples (ie neg control w/ Ab)
- Dilute secondary Ab 1:200 with TT buffer (NOT blocking buffer)
- Add 250-400ul diluted secondary Ab to each slide, incubate for 40 min. at RT, gentle shaking (prepare ABC while incubating)
6) ABC-HRP
- While secondary is incubating, prepare the ABC reagent- it must sit after mixing for at least 30-40 min, but try to make it no more than an hour ahead of time
* PBS 2.5ml
*Solution A 1 drop (biotin-avidin, 50ul), mix well, then
*Solution B 1 Drop (HRP, 50ul). Mix well
- Wash sections with TT buffer 3x 5min each.
- Add 250-400 ul of ABC-HRP to each slide. Incubate for 40 min (in humidified box) with genetle shaking
7.) DAB Reaction
-
- Wash sections once with milipore water, and prepare fresh DAB substrate from Vector Kit. You can add nickel to the substrate, which makes the peroxidiase rxn gray/black instead of brown. However, we have found that the brown stain makes it easier to distinguish real staining from the background that often associates with blood cells within tumor sections:
o ddH2O 5.0 ml
o Buffer stock 2 drops
o DAB 4 drops
o H2O2 2 drops
o Nickel (optional) 2 drops
- lay slides flat on a paper towel or bench paper, and apply DAB substrate, 250-400 ul. Let rxn run 2-10 min. away from direct light. Monitor staining in microscope. Approximate development time must be determined empirically,
- Rinse with distilled water 2-3 times, 5min each
8) Counterstain/Dehydrate/ClearMount
- Counterstain with Hematoxylin for 1sec dip in staining dish. Filter Hematoxylin through whatman paper before using
- Rinse extensively with tap water
- Dehydrate (100% EtOH x 2) and clear with Xylene (1-2 min x2) Keep in Xylene until coverslip is mounted
- Mount with permount
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