[Histonet] HELP

[Hans B Snyder]

Hello All,

Does anyone have a protocol for the Anti-Neutrophil antibody [NIMP-R14]
(ab2557) for mouse tissue?  I have been trying to extinguish the background
staining but cannot find the right combination.
​ We are doing this by hand. ​
 The recommended dilution for this antibody is 1:50 with HIER.   See used
protocol below.

So far I have tried:
1. concentrations 1:25, 1:50, 1:75, 1:100  - 1:50 gives very high
background but good positive staining, 1:100 gives low background but not
much positive staining.

2. HIER - 3, 5, 10,  20, minutes  @ 96C, 20 minutes at 60C  all give the
same results - too much back ground

3. We have tried using the H2O2 before the primary and after, increasing
the time from 30 minutes to 45 minutes.

4. Tried incubating in the primary for shorter time (20 minutes) and longer
overnight at 4C.

5. Tried blocking using 3 different serums, first each one then
combinations and longer times.

6. Tried cutting the DAB concentration in 1/2 then 1/3.


We have not tried enzyme or acid epitope retrieval yet.



*Does anyone have a working protocol or suggestions on what to try?*


​Thank you in advance.​





Procedure:

Deparaffinize

1.      Heat slides to 60C for 10 minutes (oven).

2.      Dry slides at room temp 5 minutes.

3.      Xylene 5 minutes.

4.      Xylene 5 minutes.

5.      100% ethanol 5 minutes (dehydration).

6.      100% ethanol 2 minutes (dehydration).

7.      95% ethanol for 2 minutes (rehydration).

8.      70% ethanol for 2 minutes (rehydration).

9.       Run in DI water for 10 minutes.



Antigen Retrieval

1.      Submerge in 0.01M Citrate Buffer (pH 6.0) at 96C for 5 minutes.

2.      Cold running water 5 minutes.



Staining

1.      Wash in PBS for 1 min.

2.      Permeabilize with 0.1% Triton X / 2.5% horse serum + 2.5% fetal
bovine serum for 2 hours.

3.      Incubate in primary antibody 30-45 minutes at in 2.5% horse serum
in PBS.

4.      Wash 3x in PBS/tween for 5 minutes each.

5.      Fresh 3.0% hydrogen peroxide (H2O2) 2x for 20 minutes each.

6.      Wash 3x PBS 2 minutes each.

7.      Incubate with anti-Rat Ig impress solution (according to vector’s
instructions) 45 minutes.

8.      Wash 3x in PBS for 5 minutes each.

9.      Prepare Impact DAB substrate (see insert).

10.  Using a light microscope, incubate sections with 50-100ul, until
desired darkness occurs (5-30 seconds).

11.  Stop DAB reaction using deionized water.

12.  Wash in DH2O for 5 minutes

13.  Counterstain in Harris hematoxylin for 30 seconds.

14.  Wash in DH2O for 5 minutes.

15.  Dehydrate 2x in 95% alcohol for 30 seconds each.

16.  Dehydrate 3x in 100% alcohol for 2 minutes each.

17.  Clear 2x in xylene 5 minutes each.

18.  Mount coverslips using Leica mounting media.


Hans B Snyder
Histologistics
60 Prescott Street
Worcester, MA 01605
508-308-7800
hans <@t> histologistics.com