Methods and Techniques Discussion => Immunocytochemistry (ICC) => Topic started by: innsbrucker on September 30, 2014, 06:40:31 AM

Title: Immunostaining of frozen tissue on glass slides
Post by: innsbrucker on September 30, 2014, 06:40:31 AM
Hello,

I would like to do staining with tissues on glass slides. The brain slices are 30 microns. I would be very thankful if someone can share his/her immunostaining protocol for staining on glass slides.

I am curious to know the volume of T/PBS-T and antibodies to be added. While doing staining on free floating slides i wash with 700 yl of TBS-T and add 500 yl of antibodies.

Thanks a lot :)

Cheers
Title: Re: Immunostaining of frozen tissue on glass slides
Post by: gula on October 01, 2014, 01:18:58 PM
Hi, I would recommend to cover the whole section with your antibody solution in a horizontal position of the slide. The section can be circular marked with a Pap-Pen, in a small distance to the tissue. Incubation has to be done in a moist environment, so use a "wet chamber" for incubation to inhibit drying of the tissue escpecially on the edges. This would lead to unspecific background staining.

Es soll eine regelrechte Flüssigkeitshaube entstehen.

Rinsing of the slide can be done in plenty of buffer in a coplin jar. Use everytime a fresh portion of buffer for rinsing. Move the slide gently in the liquid in order not to loose the tissue.

Der Objektträger mit dem anhaftenden Gewebeschnitt kann in einer mit Puffer gefüllten Küvette sehr einfach gespült werden.

hope this helps
Gula