I am trying to adhere the tissue in slides, after being embedded in paraffin and cut to 13 microns but when I started with my IHC protocol the tissueis detached from the slide before applying the primary antibody. It worries me greatly that my tissues are not adhering and, if I arrive, when I incubate with coverslip, this will dislodge all the tissue. Any suggestion will be welcome.
Do you follow the basics of IHC-protocols?
Usage of clean, grass-free, adhesive slides,
out of the waterbath getting rid of water beneath the section,
drying vertically to rinse off the residuent water,
baking the sections onto the glass-slides in an 60°C oven for 30-60 min,
deparaffinize in 2 or 3 batches of xylene for 5 min each and rehydrate in descending ethanol-concentrations till water or buffer,
Heat pretreatment according to your primary antibody.
There are harsher and softer ways of HIER. From cooking in boiling buffer to hot steam in an rice-steamer. A method between is incubating in a buffer at 80-90 °C (waterbath) for 30-45 min.
The problem of non-adhearing sections occurs often in Researchgate. Make a suitable search in the archives...
Thank you very much for your answer. Am using a basic IHC protocol, will try with more xylene washes and more time baking the slides.
Also what glue is on the slides, dont use gelatin it dissolves
And whatever "glue" you use they are far stickier if the slides are dried at 37oC overnight
Hi Katiria, I would recommand to use either Superfrost plus slides or, a little bit cheeper while doing it by yourself, slides subbed with a solution of 0,5 gelatine and 0,05 chromalaun. This slides are suitable also for thicker sections. Thinner paraffin sections 5-10µm will also be helpful. Good luck!
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