[Histonet] Achieving Better Antibody Penetrance in my Tissue Sections (IHC)

[Leslie]

Is there a reason you probe with the two primaries independently?  I stain rat spinal cord and brain, 35 microns thick free floating, with both primary's at the same time.  I stain at room temp on a shaker overnight.  This seems to work pretty good.

Thanks

Leslie

Sent from my iPhone

> On Jan 19, 2015, at 11:53 AM, Michael Bruno <mbruno3 <@t> buffalo.edu> wrote:
> 
> Hi All,
> 
> I'm new to graduate school and the world of immunohistochemistry.
> 
> I am looking for a way to change my protocols so that the tissue sections are stained/penetrated by the antibodies more effectively.
> 
> I am doing an anti-substance P stain, followed by an anti-RFP stain in 50um sections of rat brains. The rats were injected unilaterally with an AAV10 (with an mCherry tag) virus (into the dorsal striatum) that was designed by my professor.  When I observed the sections on a confocal microscope, it was apparent that the center of the sections do not have the same intensity of staining that is found on the outside edges of the tissue.
> 
> ==========
> 
> * Here is an outline of my procedure :
> * Take tissue sections I am interested in (previously observed for
>   native fluorescence)
> * Rinse the floating sections in ~10mL PBS 3x5 minutes
> * Rinse the floating sections in ~10mL 0.5% PBST (Triton X) 3x5 minutes
> * Block sections in 5% normal goat serum (NGS), with 0.25% PBST for 1
>   hour @ room temp
> 
> * Incubate sections in anti-substance P (anti-SP) at a [1:400] in 2.5%
>   NGS, in 0.25% PBST.  Overnight at 4*C, shaken.
> *
>     o I used a 24-well plate to hold the sections separately.  Each
>       well contained 300ul of the solution described.  I had 10
>       sections, and some controls.  So 8 sections at a time would be
>       treated with the antibodies.
> * Sections rinsed in PBS 3x5 minutes
> * Sections incubated in secondary antibody (Ab), Alexa488 [1:1000] in
>   0.25% PBST for 2 hours at room temp.
> * The the tissue sections are rinsed in PBS 4x10 minutes to remove
>   extra antibodies.
> 
> * At this point I start the second primary incubation: anti-RFP biotin
>   conjugated [1:2400] in 0.25% PBST, overnight in the cold room.
> * Rinse sections in PBS 3x5 minutes
> * Incubate the sections in the second secondary antibody, Cy5 [1:2000]
>   in 0.25% PBST for 2 hours at room temp.
> * Rinse the sections in PBS, 3x5 minutes.
> 
> * Then the sections were mounted to glass slides.  Prolong Gold w/
>   DAPI was the mounting medium.  Cover slipped.
> 
> * I dried for a few days, in a desk drawer; covered and dark.
> 
> 
> ==========
> 
> Thanks for reading all of this.  Since it would be impossible to change the thickness of the sections (they are cut and stored in the fridge in 0.02% sodium azide), I think I need to change incubation times, temperature, and PBST concentrations.
> 
> Any tips would be greatly appreciated!  If you have had a similar issue in the past, I would like to know what you did to fix it.
> 
> Thanks!
> Mike
> 
> 
> 
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