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

[koellingr <@t> comcast.net]

Michael, 
Not sure from your explanation if your mCherry tag is less intense on the inside of sections or if you are referring to the P and RFP Alexa and Cy5 stain intensity????  If the former, not sure about that.  If the later, having done very similar thing in grad school, but with different tissue and molecule targets and different antibodies, your protocol looks very similar to what I did for years.  With exception that I extended those secondaries time-wise.  Whether right or wrong, my thinking that if it took 'overnight/shaken" for a 150kDa antibody to penetrate through 50u (which is what I used), it would take more than just 2 hours for an even bigger molecule (Ab+tag) to penetrate.  I'd try extending secondary times while keeping everything else same and look for better penetration.  Otherwise, our protocols would look nearly identical. 
  
Ray Koelling 
Lake Forest Park, WA 

----- Original Message -----

From: "Michael Bruno" <mbruno3 <@t> buffalo.edu> 
To: "histonet <@t> lists.utsouthwestern.edu >> Histonet Listserve" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Monday, January 19, 2015 11:53:01 AM 
Subject: [Histonet] Achieving Better Antibody Penetrance in my Tissue        Sections (IHC) 

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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