Hallo Syeda, it depends on the Kind of section you use (Paraffin or frozen sections), of the thickness of your sections and the immunhistochemistry protocol you like to use. For 5µm Paraffin sections or 30µm PFA fixied and kryoprotected frozen section you can use for the blocking step 10% normal Serum in 0,2%Triton-X100(Tx) in PBS/TBS plus 20% Avidin-block (if you use streptavidin or ABC as detection system). Dilution medium for the primary antiboedy: 1% normal-Serum in 0,2%TX-PBS/TBS plus 20% Biotin-Blocking (Streptavidin or ABC). secondary antibody and detection System like Streptavidin or ABC: 1% n-Serum in 0,1%Tx.PS/TBS. Good luck!

0.1% TX10 is a standard concentration of detergent. You use higher concentration for sub/extra cellular structures, which are difficult to penetrate, e.g., synapses (0.5%). Frozen sections require more gentle treatment and less detergent. To reduce background, you can preblock for longer than 30min-1h recommended. Use 5-10% normal serum (good source: Jackson immunoresearch, they have superb explanations, too). for 2ndary antibodies use same species as blocking serum (e.g., if you use normal goat serum, then use goat-anti-mouse, goat anti-rabbit etc. antibodies). Frequently using 2ndary antibodies from other species does not cause any change in background, sometimes it does. To further reduce background, don't overfix the tissue, the aldehyde groups on tissue cause substantial increase in background. To deal with overfixing, you can wash for longer time if you use PFA (it is partially reversible crosslinker). You can also use glycine (0.3M, search protocols) to suppress aldehyde-caused autofluorescence. Also use 2ndary antibodies, which are correctly diluted (w/o clumps) and not too old. To solve this question about whether my 2nday ABs are too old, store them in 50% glycerol at -20C (rather as an aqueous solution at +4C (then they will be good for 10+ years without major change in affinity). Don't let your sections dry when doing primary or secondary antibodies. Use optimized concentration of primary and secondary ABs. Hope it helps, good luck

Dear Dr. Syeda,

Greetings,

For free floating 50-µm thickness PFA fixed brain sections, we use 0.3 % Triton X-100

Dear All,

Many thanks for sharing your protocols. Its really very helpful for me. Stay Happy and blessed!

Ahmed Saad Ahmed HASSANEEN Igor O. Nasonkin Mohamed Mahdy Ute Neubacher

Thank you once again :)