ࡱ> .0- Xbjbj,(,( *NBNB||||| ,!!!!!!,M!|M||n||rWTÄNe7j 02DD| MMD B:   Immunocytochemistry on Paraffin Sections (ICC) DAY 1 Deparaffinize sections. Xylene #1 5 minutes Xylene #2 5 minutes Xylene #3 5 minutes 100% EtOH #1 3 minutes 100% EtOH #2 3 minutes 100% EtOH #3 3 minutes 95% EtOH 3 minutes 70% EtOH 3 minutes Quick rinse PBS. 5 minute rinse PBS. Carefully wipe slide around sections. Outline sections with hydrophobic pen. Do only 2 slides at a time so sections dont dry out. Sections always need to be kept moist. Incubate slides in 4% Normal Serum for 20 minutes in humid chamber. (Use blocking serum from species secondary antibody is made in; for example for a mouse monoclonal primary antibody detected with a horse anti mouse secondary antibody, block in horse serum.) Blot off Normal Serum. Only do 2 slides at a time. Incubate with primary antibody overnight at 4oC in humid chamber. *Do a section with no primary antibody to test what the background will be with fluorescent secondary antibody only. DAY 2 Quick rinse PBS. 5 minute rinse PBS. Incubate with Secondary Antibody 1 hour at room temperature in dark, humid chamber. All following steps need to be done in the dark. Alexa Fluor secondary antibody. Choose appropriate antibody to detect primary antibody. Dilute 1:300 in TAB. Quick rinse PBS. 5 minute rinse PBS. 2-3 quick rinses ddH2O. Coverslip with FluorSave Reagent (Calbiochem Catalog # 345789). Store slides in the dark. Solutions for Fluorescence ICC PBS Make 1L 0.1M NaH2PO4 (monobasic) by adding 13.8g NaH2PO4 to about 950ml ddH2O. q.s. volume to 1L. Make 1L 0.1M Na2HPO4 (dibasic) by adding 14.2g Na2HPO4 into about 950ml Add 9g NaCl to the 1L of dibasic sodium phosphate. Stir well to dissolve. Monobasic sodium phosphate to adjust pH to 7.4. Use ddH2O. q.s. volume to 1L. Recheck pH. To about 900ml ddH2O, add 13.22g Tris HCl + 1.94g Tris Base. Adjust pH to 7.4. Stir. Adjust final volume to 1L with ddH2O PBS/BSA Solution(Tris/Bovine Serum Albumin) To 250ml Tris Buffer solution add 1.25g BSA (warm BSA to room temperature before weighing). Stir to dissolve. Adjust pH to 7.4. PAB Solution (Tris Azide BSA) 50ml PBS/BSA + 500ml 10% Sodium Azide in ddH2O. 4% Normal Horse Serum 0.5ml Normal Horse Serum + 12ml TAB /7 8 D u   89<= opXhO:h>CJH*OJQJaJhO:h>5CJOJQJaJhO:h>CJH*OJQJaJhO:h>CJOJQJaJhO:h>>*CJOJQJaJ&/08Pez$ L u # ~  & Fgd>h^h & F~ PrsBDVX & Fh^hh^hgd> & Fgd> & F(/ =!"#$% 666666666vvvvvvvvv666666>6666666666666666666666666666666666666666666666666hH6666666666666666666666666666666666666666666666666666666666666666666666666662 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ OJPJQJ_HmH nH sH tH <`< NormalCJ_HmH sH tH DA D Default Paragraph FontViV  Table Normal :V 44 la (k (No List PK!pO[Content_Types].xmlj0Eжr(΢]yl#!MB;.n̨̽\A1&ҫ QWKvUbOX#&1`RT9<l#$>r `С-;c=1g'}ʅ$I1Ê9cY<;*v7'aE\h>=,*8;*4?±ԉoAߤ>82*<")QHxK |]Zz)ӁMSm@\&>!7;ɱʋ3װ1OC5VD Xa?p S4[NS28;Y[꫙,T1|n;+/ʕj\\,E:! t4.T̡ e1 }; [z^pl@ok0e g@GGHPXNT,مde|*YdT\Y䀰+(T7$ow2缂#G֛ʥ?q NK-/M,WgxFV/FQⷶO&ecx\QLW@H!+{[|{!KAi `cm2iU|Y+ ި [[vxrNE3pmR =Y04,!&0+WC܃@oOS2'Sٮ05$ɤ]pm3Ft GɄ-!y"ӉV . `עv,O.%вKasSƭvMz`3{9+e@eՔLy7W_XtlPK! ѐ'theme/theme/_rels/themeManager.xml.relsM 0wooӺ&݈Э5 6?$Q ,.aic21h:qm@RN;d`o7gK(M&$R(.1r'JЊT8V"AȻHu}|$b{P8g/]QAsم(#L[PK-!pO[Content_Types].xmlPK-!֧6 -_rels/.relsPK-!kytheme/theme/themeManager.xmlPK-!!Z!theme/theme/theme1.xmlPK-! ѐ'( theme/theme/_rels/themeManager.xml.relsPK]# X ~ X 8Euz2;EOW[(,045:`eu2{@o::::::::   q_XJ@B@UnknownG*Ax Times New Roman5Symbol3 *Cx Arial3TimesA$BCambria Math"AhT&r&KaKa24a@HP(?2!xx ,.Immunocytochemistry on Paraffin Sections (ICC) Diana Lurie CSFN U of M     Oh+'0 $0 T ` l x'0Immunocytochemistry on Paraffin Sections (ICC) Diana Lurie Normal.dotm CSFN U of M5Microsoft Macintosh Word@^в@//@XwY@r.e7Ka ՜.+,0, hp  'ϻ /Immunocytochemistry on Paraffin Sections (ICC) Title  !"#$&'()*+,/Root Entry FpNe711Table `WordDocument*SummaryInformation(DocumentSummaryInformation8%CompObj` F Microsoft Word 97-2004 DocumentNB6WWord.Document.8