You should not keep paraffin sections in 4C to avoid this exact problem. I suggest using a hot air blower on slides until paraffin becomes slightly melted (more transparent) that will ensure the reattachment of your samples to the glass. I had experience with 30 years old paraffin sections stored at RT and still got good immunofluorescence signals.

FFPE slides do not need to be kept at 4C. We have stored slides for years in a cool, dry environment. As long as you 'bake' the tissue on after sectioning; clinically we use a 60C oven for 30 min, they can keep years.

One of the factors that degrades slides over time is moisture, keeping slides at 4C is likely to induce some condensation on the slides which can lead to sections coming off slides, or worse, mold.

I agree with Anton Lennikov , warm them and allow the paraffin to soften and any residual water trapped to evaporate.

And use new diluent, 8 months is a bit much.

Anton Lennikov Jason Rogers - Thank you very much! I'll try heating the slides and getting fresh diluent and see if that helps. Do you think it would be best to just cut new sections though? Luckily I kept my blocks at RT. Also do you store your slides/blocks with desiccant? And do you recommend a different diluent? That one has worked well for me but it would be nice not to have to buy it pre-made. I'm doing immunofluorescent staining mostly and sometimes DAB if that makes a difference. Thanks again!

It is preferable to store slides with the desiccant but we have never done it. As I said to my surprise the 30 years old PFA/Paraffin sections were quite efficiently stained after being stored in RT. You can of course section the blocks again, but reheating the existing sections would probably be easier.

We always used a PBS/BSA/Azide diluent we made up that week, had a liter of the stuff on ice and just thawed it out 50 mL at a time. Your mileage may vary, but looking at what IHC world charges (owch) I would seriously consider looking at making up your own.

I have stored slides vacuum sealed with a desiccant, but those were cut frozen slides and had to be kept in the freezer, never needed it with FFPE slides.

I would only say you would need to consider some sort of desiccant if you end up having to warehouse slides offsite and you don't know the conditions they are kept.

Jason Rogers Perfect thanks! Sorry one more question - I was using that diluent because it claims to remove the necessity for blocking and it may because I never had a problem with non-specific secondary signal. Do you normally block in serum/BSA solution before applying antibody when you use the homemade diluent? I've heard mixed things about whether it was really necessary but perhaps it depends on the tissue.

Yes, block with normal serum before applying antibodies. The newest gen of antibodies probably don't need an additional blocking step, but I would directly test that before assuming anything.

I saw that product say that you don't need to serum block, but it doesn't say why or how and I distrust that kind of labeling. If it is the best for you, by all means use it, just giving you options.

Just heat the slide befor work on hot plate and then cooled it at R.T.