IMMUNOPEROXIDASE STAINING-BASIC
Paraffin Sections:deparaffinize and hydrate to water.
4-4 minute changes of Xylene
2-3 minhute changes of 100% EtoH
1-3 minute change each of 95% & 70% EtoH
several rinses with tap water then distilled water
Frozen sections: general: 5 minutes in PBS to remove OCT then up and down in
graduated alcohols (70, 95, 100, 95, 70%-1 minute each), PBS 5 minutes.
Check protocol for pretreatments if any. There are various pretreatments for different
antibodies.
Dry slides around sections and isolate with PAP pen to contain solutions.
Block for 10 minutes, Room Temperature (R.T.)37°C, aspirate solution off.
Primary AB: most at room temperature for 1:15 hour, BrdU for 1:30 hour.
Overnight at 4°C use overnight coverslips
calculate 250ul/slide, dispense 225ul; 80ul/section dispense 70ul
most antibodies use Triton-100 (Tx-100) at 0.5 %.
Washing solution: 3x5 minutes, agitate first 30 seconds. Place second bath on
shaker. After completing, discard #1 wash and rotate 2 & 3 forward; place fresh wash
in 1st jar making it the 3rd.
Secondary Antibody: 1 hour R.T. Calculate 225ul/slide, dispense 200ul;
80ul/section, dispense 70ul
1:50 bHAM+1:50 NRS (normal rat serum)+ Horse Block
1:150 bGAR+1:50 NRS (normal rat serum)+ Goat Block
1:150 bRAG+1:50 NRS (normal rat serum)+Rabbit Block
**take care to avoid mixing goat & rabbit even when washing**
Wash 3x5 minutes
Tertitary Antibody: 45 minutes R.T.
For DAB: Avidin–biotinylated Horseradish Peroxidase (* denotes Elite
kit)
1:50 Avidin +
(to make things easier, prepare after you are finished applying
secondary.)
Leave at R.T.
Fluorescent staining: Made up in the base solution:
1:200 Fluoricein Strep-Avidin(F Strep Av, F St Av)
1:200 AMCA Strep Avidin (AMCA St Av)
Wash 3x5 minutes
DAB: Add 30% hydrogen peroxide: 67ul/200 ml;16.8ul/50 ml. Agitate for 30 seconds,
place on shaker. Leave in solution for a total of 8
minutes. Place in tap water for 23 minutes then in several changes
of water then in running distilled water for 3-5
minutes. DAB
solution is disposed of in appropriate container in the hood, first
rinse water is
then rinsed thoroughly.
Dehydrate through alcohols, 70%, 95%, 100% x2 and clear with xylene x3, about 30
seconds agitation per dish .
coverslip with DPX
SOLUTIONS
BASE SOLUTION(4%5%): 4% BSA (Bovine Serum Albumin)in PBS with 5% Non
BLOCKING SOLUTION: made with appropriate normal serum and Base
concentration: 1 ml serum + 9 mls Base
Solution.10%
WASH SOLUTION(buffer): 0.5% BSA in PBS. 1L for staining dishes(900 ml
distilled water + 100 ml of 100X PBS{in freezer} + 5 g BSA) 250 ml for coplin
jars.
DAB: (diaminobenzidine tetrahydrochloride): 2mg/ml (carcinogen, use proper
precautions)
50 ml/coplin jar:1.47g sodium citrate/50ml Double distilled water,
200 ml/staining dish: 5.88g sodium citrate/200ml dd water
-pH between 5.05-5.15 with HCl,
- add DAB, mix well, vaccum filter with P2 filter paper.
-Just before use, add 30% Hyrogen preoxide:
16.8µl hydrogen peroxide/50ml
67µl hydrogen peroxide/200ml
Label and date ALL solutions. 4% BSA and serums should be kept frozen except when
in use.
Primary antibodies, secondaries and tertiaries should be dated (month and year) when
Fat Dry Milk(N
they are put into general use from storage. Let’s try to avoid duplicates.
Clean up spilled materials and put things away after use: chemicals back to cupboards,
staining chambers to cupboards, pipetors to racks, instruments to drawers. Counters
need to be wiped after use and/or spills.
DAB glassware should be bleached and work area cleaned up after use. Rinse
glassware and dispose of tips, vials, corks and filter paper the next day, DO NOT
LEAVE FOR THE PERSON WASHING DISHES.
Block for 10 minutes, Room Temperature (R.T.)37°C, aspirate solution off.
Primary AB: most at room temperature for 1:15 hour, BrdU for 1:30 hour.
calculate 250ul/slide, dispense 225ul; 80ul/section dispense 70ul
most antibodies use Triton-100 (Tx-100) at 0.5 %.
Washing solution: 3x5 minutes, agitate first 30 seconds. Place second bath on
shaker. After completing, discard #1 wash and rotate2 & 3 forward; place fresh wash
in 1st jar making it the 3rd.
Secondary Antibody: 1 hour R.T. Calculate 225ul/slide, dispense 200ul;
80ul/section,dispense 70ul
1:50 bHAM+1:50 NRS (normal rat serum)+ Horse Block
1:150 bGAR+1:50 NRS (normal rat serum)+ Goat Block
1:150 bRAG+1:50 NRS (normal rat serum)+Rabbit Block
washing**
Wash 3x5 minutes
Tertitary Antibody: 45 minutes R.T.
For DAB: Avidin–biotinylated Horseradish Peroxidase (* denotes Elite
(to make things easier, prepare after you are finished applying
For Fluorescent staining: Made up in the base solution prior to use:
1:200 Fluoricein Strep-Avidin(F Strep Av, F St Av)
1:200 AMCA Strep Avidin (AMCA St Av)
Wash 3x5 minutes
kit)
1:50 Av
secondary.) L
DAB: Add 30% hydrogen peroxide: 67ul/200 ml;16.8ul/50 ml. Agitate for 30 seconds,
place on shaker. Leave in solution for a total of 8
minutes. Place in tap water for 23 minutes then in several changes
of water then in running distilled water for 3-5
minutes. DAB
solution is disposed of in appropriate container in the hood, first
rinse water is
then rinsed thoroughly.