[Histonet] p2y12 cryo rat problem high background

[Allyse Mazzarelli]

Hi Esther,

I worked extensively with brain and spinal cord sections in the past, in
multiple species. You need endogenous quenching step.

I would always make my own (commerically available components yielded
different results). I used a 0.09% H2O2 in 6% Triton-X 100: (formula: 200mL
1x PBS + 6mL H2O2 + 0.33mL 6% Triton-X 100 solution).

Are you samples fixed frozen or fresh frozen? If fixed-frozen, there is no
need for the acetone step. In fact, acetone in brain and spinal cord
sections makes the tissue brittle. I would recommend this is skipped.

If your samples are fresh-frozen, I would recommend a 95% EtOH fix. This is
significantly gentler on the tissue.

I have no experience with the Envision system, but the polymer system I
would always use was from Cell Signaling Technologies. What antibody are
you using?

On Thu, Nov 15, 2018 at 7:48 AM Kooijman, E.J.M. (Esther) via Histonet <
histonet at lists.utsouthwestern.edu> wrote:

> Hello Bobbie,
>
> But should I elimination endogenous peroxidase activity in brain/spinal
> cord tissues?
> Brains and spinal cord was harvested after cervical dislocation, then the
> tissue was snap frozen (isopentane..).
>
> thanks for your help,
> Esther
>
> -----Oorspronkelijk bericht-----
> Van: Boyce, Bobbie [mailto:Bobbie.Boyce at nemours.org]
> Verzonden: donderdag 15 november 2018 12:21
> Aan: Kooijman, E.J.M. (Esther)
> Onderwerp: RE: p2y12 cryo rat problem high background
>
> Hi Ester,
> Try Peroxoblock (Zymed) before your BSA block, but you have to be careful
> not to leave it on too long or it will eat your tissue. It's been a while
> since I've had to used it.
>
>
> Bobbie Boyce
> Histology Specialist III
> DuPont Experimental Station
> Nemours- Biomedical Research Department
> Histochemistry and Tissue Processing Core
> 200 Powder Mill Road, Bldg.400  Rm.5240
> Wilmington, DE 19803
>
> (lab) 302-651-6771
> (fax) 302-651-5010
>
>
>
> -----Original Message-----
> From: Kooijman, E.J.M. (Esther) via Histonet <
> histonet at lists.utsouthwestern.edu>
> Sent: Thursday, November 15, 2018 5:53 AM
> To: histonet at lists.utsouthwestern.edu
> Subject: [Histonet] p2y12 cryo rat problem high background
>
>  **This is an External Email -  Please DO NOT open attachments or click
> links from unknown senders or unexpected email. **
>
> Hello all,
>
>
>
> I am trying to stain cryo brain sections from the rat 7um but having a lot
> of background. What am I doing wrong. Below the protocol is used and tried
> to adjust…
>
>
>
> 1-      Fix the tissue with cold acetone (-20 ⁰C) for 10 minutes
>
>
>
> 2-      Let the slide dry for 30 minutes at room temperature. Isolate the
> sections with DAKO pen
>
>
>
> 3-      Block sections with BSA 2% in PBS for 1 hour at room temperature
>
>
>
> 4-      Add primary antibody in PBS/BSA 1%, overnight at 4⁰C or 1h at room
> temperature
>
>
>
> 5-      Wash 3x5 minutes with PBS/tween-20 0.05%
>
>
>
> 6-      Add 100 µL Envision solution (goat anti-mouse/rabbit) and incubate
> for 1hr at RT
>
>
>
> 7-      Wash 3x 5 minutes with PBS/tween-20 0.05%
>
>
>
> 8-      Incubate with DAB (1:50) for 10 min (between 5-10 min; check color
> development)
>
> (wear gloves, carcinogenic!)
>
>
>
> 9-      Rinse thoroughly with miliQ water
>
>
>
> 10-   Stain with haematoxylin for 1 min
>
>
>
> 11-   Wash with running tap water for 5 min
>
>
>
> 12-   Start the alcohol/xylene series (70% EtOH -> 80% EtOH -> 96% EtOH ->
> 100% EtOH -> 100% EtOH/xylene -> xylene -> xylene)
>
>
>
> 13-   Mount slides with entallan
>
> Kind regards,
>
>
>
>
>
> Esther Kooijman  |  Research Technician  |  Department of Radiology and
> Nuclear medicine
>
> The Netherlands
>
>
>
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