Hello,
I'm doing immunofluorescence staining, under confocal microscopy, on 30um thick hippocampal tissue from rats. The fresh brains were post-fixed in formaldehyde, rinsed and are currently stored in 30% sucrose with azide. I plan to cryosection the tissue at 30um thickness and antibody stain for microglia and astrocytes using anti-Iba1 and anti-GFAP, respectively.
To confirm amyloid formation, I will co-stain with Thioflavin T.
Can someone kindly assist me with the following:
1) How to make stock solution of Thioflavin T?
2) How do I dilute the stock Thioflavin to a final working concentration of 20Mm?
3) How long should the sections be incubated in ThT before I can rinse?
4) I use TBS as a wash buffer, will it be suitable to rinse sections in TBS following incubation in ThT? Or do I have to rinse with ethanol?
Thank you in advance for your assistance.
Hallo Nonkululeko,
If you like to use the Thioflavin-staining as a counterstain for your Glia-IHC I would recommand to mount the sections on gelatine coated slides after last antibody treatment. Let them air dry over night. Then rinse the slides several times in destilled water, stain them for 3 min in a 1% Thioflavin solution in water, rinse again several times in destilled water, differnciate for 10-20 min in 1% acetic acid, final rinse in runnign tap water, cover slipping. This Protocol (Vassar & Culling 1959) is used for paraffin sections. I recommand to check the optimal staining and differenciation time with positiv Amyloid beta tissue without immuno, because you should control your staining result under the microscope.
Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue