{"id":2294,"date":"2017-06-08T20:53:52","date_gmt":"2017-06-09T00:53:52","guid":{"rendered":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/if\/"},"modified":"2018-07-20T16:43:24","modified_gmt":"2018-07-20T20:43:24","slug":"if","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/if\/","title":{"rendered":"Paula’s Immunofluorescence Protocol"},"content":{"rendered":"
\n

A Dohlman Lab Protocol<\/em><\/p>\n

\n

Day 1<\/h4>\n

Inoculate 25-50ml from a saturated starter culture. Grow overnight to OD600nm= 0.4-0.8
\n
<\/p>\n

Day 2<\/h4>\n

Slide Preparation:<\/p>\n

-Coat slides with 20 ul of 1 mg\/ml Poly-L-Lys sol\u2019n. Incubate for 5 min.<\/p>\n

-Aspirate sol\u2019n and air dry<\/p>\n

-Wash slide in 50 ml falcon tube with ddH20 for 30 min.<\/p>\n

-Let slide air dry<\/p>\n

Harvest 5 OD\u2019s of cells by vacuum filtration<\/p>\n

Resuspend in 1 ml the same media + 1 ml 2X Fixative Solution.<\/p>\n

Fix for 1 h at RT with gentle shaking<\/p>\n

Harvest cells @ 3,000 rpm 5 min RT and discard supe in hazardous waste container<\/p>\n

Wash cells 2x with 5 ml NaPi\/Sorb. Buffer via vacuum filtration<\/p>\n

Resuspend cells in 1 ml NaPi\/Sorb. Buffer + 10 ul 14.3 M 2-ME<\/p>\n

+ 10 ul of 5 mg\/ml (in NaPi\/Sorbitol Buffer) Zymolyase 100T<\/p>\n

Incubate for 5-15 min @ 30\u0192 C with gentle shaking to spheroplast cells<\/p>\n

They are done when they lyse in water under the microscope.<\/p>\n

Harvest cells 2,000 rpm, 3 min, 4\u00baC<\/p>\n

Wash cells 3x GENTLY with 5 ml NaPi\/Sorb. Buffer<\/p>\n

Resuspend in 250-500 ul NaPi\/Sorb. Buffer<\/p>\n

Apply 20 ul cells to wells for 15 min.<\/p>\n

Wash 3x with 50 ul PBS\/BSA Wash Buffer<\/p>\n

Rinse using an eppendorf repipettor and an aspirator, or a glass washing chamber, or 50 ml falcon tube.<\/p>\n

Permeabilize cells with P Buffer for 10 min.<\/p>\n

Wash cells 10x with 50 ul PBS\/BSA Wash Buffer<\/p>\n

Block cells 30 min with PBS\/BSA Wash Buffer in dark humid chamber<\/p>\n

Wash 5x with 50 ul PBS\/BSA Wash Buffer<\/p>\n

Incubate cells with 30 ul of 1\u00ba Ab in PBS\/BSA Wash Buffer for 1 h, RT in dark moist chamber<\/p>\n

= HA mouse Ab- 1:2000<\/p>\n

= GFP rabbit Ab- 1:250<\/p>\n

Wash cells 10x with 50 ul PBS\/BSA Wash Buffer<\/p>\n

Incubate cells with 30 ul of 2\u00ba Ab in PBS\/BSA Wash Buffer for 1 h, RT in dark moist chamber<\/p>\n

= GoatAntiRabbit Cy3-conjugated 1:500<\/p>\n

= GoatAntiMouse Cy3-conjugated:500<\/p>\n

Wash cells 7x with 50 ul PBS\/BSA Wash Buffer and 3x with 50 ul PBS<\/p>\n

Rinse using an eppendorf repipettor and an aspirator.<\/p>\n

Mount with fluoromount G and seal with nail polish
\n
<\/p>\n

Solutions and Buffers<\/h4>\n

2X Fixative pH 6.5 (sterile filtered)<\/p>\n

2 % glucose 5 ml 40 % glucose<\/p>\n

40 mM EGTA 10 ml 0.4 M EGTA<\/p>\n

7.4 % formaldehyde 20 ml 37 % stock<\/p>\n

in 2 X PBS 20 ml 10 X PBS<\/p>\n

dH20 to 100 ml<\/p>\n

NaPi\/Sorbitol Buffer pH 6.6 (sterile filtered)<\/p>\n

100 mM NaPi, pH 6.6 25ml 1 M NaPi pH 6.6<\/p>\n

1.2 M sorbitol 75 ml 4 M Sorbitol<\/p>\n

dH20 to 250 ml<\/p>\n

PBS\/BSA Wash Buffer (sterile filtered)<\/p>\n

10 mg\/ml BSA 1g BSA<\/p>\n

100 ml 1 X PBS<\/p>\n

P Buffer<\/p>\n

10 mg\/ml BSA 1 g BSA<\/p>\n

0.5 % SDS 5 ml 10 % SDS<\/p>\n

100 ml 1 X PBS<\/p>\n

PBS Buffer<\/p>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"

A Dohlman Lab Protocol Day 1 Inoculate 25-50ml from a saturated starter culture. Grow overnight to OD600nm= 0.4-0.8 Day 2 Slide Preparation: -Coat slides with 20 ul of 1 mg\/ml Poly-L-Lys sol\u2019n. Incubate for 5 min. -Aspirate sol\u2019n and air dry -Wash slide in 50 ml falcon tube with ddH20 for 30 min. -Let slide … Read more<\/a><\/p>\n","protected":false},"author":22429,"featured_media":0,"parent":2239,"menu_order":15,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2294","page","type-page","status-publish","hentry","odd"],"acf":[],"_links_to":[],"_links_to_target":[],"_links":{"self":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2294","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/users\/22429"}],"replies":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/comments?post=2294"}],"version-history":[{"count":0,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2294\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2239"}],"wp:attachment":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/media?parent=2294"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}