{"id":282,"date":"2023-07-07T12:44:59","date_gmt":"2023-07-07T04:44:59","guid":{"rendered":"http:\/\/yuhongtaolab.com\/?post_type=resource&p=282"},"modified":"2023-07-07T12:44:59","modified_gmt":"2023-07-07T04:44:59","slug":"if-staining-protocol","status":"publish","type":"resource","link":"http:\/\/yuhongtaolab.com\/en\/resource\/if-staining-protocol\/","title":{"rendered":"IF staining protocol"},"content":{"rendered":"

Materials<\/strong>
\n– 4% PFA: dissolve 0.4g PFA powder in 10ml PBS, heating to help dissolve;
\n– Permeabilization buffer: 0.2% Triton X-100 in PBS;
\n– Blocking buffer: 3% BSA, 0.1% Triton X-100 in PBS;
\n– Washing buffer: 0.1% Triton X-100 in PBS;<\/p>\n

Procedure<\/strong>
\n1. Place cover slides in 24-well plate, coat with Matrigel;
\n2. Seed cells onto cover slides in 24-well plate, incubate for at least 24h to let cell attach and grow on the slides;
\n3. Aspirate medium, wash once with PBS, fix with 4%PFA, RT 10-15min;
\n4. Wash with PBS, at least two times (the cover slides can be store at 4\u2103 for up to 1 week);
\n5. Incubate in permeabilization buffer (0.2% PBST), 10min @RT;
\n6. Incubate in blocking buffer (3% PBSA), 30min @RT;
\n7. Incubate with primary antibodies diluted in blocking buffer, 2h @RT or 4\u2103 O\/N;
\n8. Wash 3x5min with washing buffer;
\n9. Stain with DAPI, 1min @RT;
\n10. Mount the cover slides on a glass slide with Vectashield or prolong gold mounting medium, seal with nail polish, air dry for 15-30min to let the nail polish solidify completely.
\n11. store glass slides at 4\u2103 for short term and -20\u2103 for long term.<\/p>","protected":false},"featured_media":0,"template":"","class_list":["post-282","resource","type-resource","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"http:\/\/yuhongtaolab.com\/en\/wp-json\/wp\/v2\/resource\/282","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/yuhongtaolab.com\/en\/wp-json\/wp\/v2\/resource"}],"about":[{"href":"http:\/\/yuhongtaolab.com\/en\/wp-json\/wp\/v2\/types\/resource"}],"wp:attachment":[{"href":"http:\/\/yuhongtaolab.com\/en\/wp-json\/wp\/v2\/media?parent=282"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}