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Author Topic: HELP. unsure of result of experiment  (Read 2410 times)

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Offline yatimms

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HELP. unsure of result of experiment
« on: April 23, 2015, 05:07:37 AM »

I have been doing IHC for a while now and my background is really high. I am not sure what's the cause of the high background. The steps below is my protocol:

•   0.01M Sodium citrate: tri-sodium citrate dihydrate. 2.94g adjust to pH6 top up to 1L mili-Q water and add 0.05% (0.5mL) tween 20
•   0.06% H2O2: 1:50 dilution with water/TBS
•   Normal blocking serum: 50uL of normal horse serum in 5mL of PBS
•   Biotinylated secondary antibody: 100uL of normal serum stock : 100uL antibody stock: 5mL buffer
•   ABC reagent: 1 reagent A: 1 reagent B : 5 PBS buffer.  *Preincubate ABC reagent 30min prior to use!
•   DAB: 30uL of chromogen to 1mL of diluents. mix well. Can be reused within 14days kept in 4deg. Deactivate it with chlorox in sealed container overnight. Discard properly. (carcinogen)

day 2
1.   Let slides air-dry overnight after cutting and fixing onto slide
2.   Bake slides at 54deg to remove excess water for 1hour
3.   Rehydrate:
•   Histoclear 10min 2x
•   100% EtOH 5min 2x
•   95% EtOH 5min
•   80% EtOH 5min
•   70% EtOH 5min
•   Rinse well with water. (do not let slides dry at any point after this step)
4.   Antigen retrieval to unmask antigen after fixing: Put slides into orange container, with sodium citrate to the brim. Add distilled water to bottom of pressure cooker to the level marked.  Press “on” <|> and loosen the knob on top of the cooker. It will auto off by 12-15min. switch off main plug. let the slides cool down for few mins rtp
5.   Wash with PBS 3x5mins *gentle agitation for all washing steps
6.   To remove endogenous peroxide: incubate 20min in 0.6% H2O2 (make fresh) *this step can be done before or after primary Ab incubation.  (discard properly)
7.   Wash with 0.2% Tween 20 in TBS 3x5mins
8.   Block 30mins with normal serum (vector) and 2% BSA
9.   Incubate with primary antibody in blocking buffer overnight 4deg. (do not use same species as sample. To be safe, use rabbit)
day 3
10.   Wash with 0.2% Tween 20 in TBS 3x5mins
11.   Incubate with secondary antibody 30min to 1hr rtp
12.   Wash with 0.2% Tween 20 in TBS 3x5mins
13.   Incubate with ABC reagent 30min to 1hr rtp
14.   Wash with 0.2% Tween 20 in TBS 3x5mins
15.   Wash 10min in TBS
16.   Incubate with DAB rtp for 30sec, look under microscope for staining. Incubate longer for low signals.
17.   Wash with water 3x5min
18.   Conterstain with hematoxylin:  Submerge sample with hematoxylin for 5-45secs
19.   Dehydrate (opp steps of rehydrate)
20.   Mount

I've attached photo of my supposed positive.  I've repeated it twice and those were the result.

The results were very inconsistent.

can someone please help me with tips.


HELP. unsure of result of experiment
« on: April 23, 2015, 05:07:37 AM »

Offline gula

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Re: HELP. unsure of result of experiment
« Reply #1 on: April 25, 2015, 03:05:20 AM »
Have you done several titrations to find out the correct titer (concentration) of the primary antibody?
Are you sure, that the AR is suitable for your samples? Fixation time? Maybe too harsh AR?
Are you sure, that deparaffination in Histoclear is done properly? fresh reagens? change to xylene?
Do you work with positive and negative controls? Does the negative control show the same background?
species of sample? species of primary and secondary antibody? blocking serum? are they compatible?

... a few questions to think about....

Re: HELP. unsure of result of experiment
« Reply #1 on: April 25, 2015, 03:05:20 AM »