albumini~edl/lO i v e r CIHRT Exhibit P-2156 Page 1 y G u l l 7 : T e r r g e 2 5 s o u r c e 14 P a a l t h - o l u m e r n H e V E a s t e Day I s : S l i d e Prepare H d e . Cut sections @ 3 microns on clear glass s 58-60 degree C. incubator overnight ' i n -' Place ,Day I I a 37 degree c.in~tl!l)lf. n - Place 200 mIl/H) PBS i g m hyps~n. 0 . 2 . o u t W e i g h - P B S : u p - Make
4 e ! l i . t ~ ~ h ~ ~ ~ o . ~ r i . i t i a t ~ n . a ' · · · · ; · ( 1 ~ ~1' ) i · , c o y e r 5 ~ ~ t . h · : { 1 : ~ a ~ 1 ~ 0 3 ~ ~ · : ' : . : : ~ < - : ~ i t ~ i b q y { Q ~ ~ . · · or30IJlinlltes/i~a.huntidificaJiQii · ¢~~~er. slide~~z:efll.lIywhh iIlopas{aibtinu.nize~)using. .·~X(18),preparedi luti~n~stpiows: 'lor20~licl~{/<i> ' ~ : · e r CIHRT Exhibit P-2156 Page 2 G u l l i v 5 8 T e r r y P a g e 2 u r c e : m e 14 h - s o V o l u H e a l t a s t e r n E . :<'. - - 2 " , - - ' " /' 5 min. Abs. alcohol ... ( 8 ) m i r i . 5 Chloroform (9) /5 min. (10) Acetone / / / min; ~ .... 5 ........ H 2 0 a p (11) Cold /// " j miliimum 5 m i n. . B S / P uminized (12) . d Y ' t i b o A n Primary Prepare dilutions of primarYantib()dYUSi.ngaIhl!llJliWi?l~>~\·· (13) d e . 0 - 200' ul per s l i 1 4 >(14)' Prepare.slides>ollebycine by . drYing()~t~ideandleavingi d :lhe>sectioll (ROArK n a r o U . P B s . tilrnof . the ,alhuiiliniji,ed · . '. ) ; . : : : ; E C T I(jl(S.DR~! s <BUBBLE 9)':( 110#ci'1'I.ETT!l~ . . . .' .': • . . . • . • . . . . • . ...•. . . . • . . . . . . · . . • . . . i<. .•..... . i{ time ) f . . :. each' . Wash 1 6 ) . · · · · ( .'. . .' asqueezeholUe;" of the albilrninill'.edPBS .. s h a staining d i o . . .' . (l7)'l'ransfer t " . • Antibody . ;)~ecandarY > . :..•..... : . " '. ..... ,; ... :.::..:::: .. .. : . . . :::.: .........•........•...•.•..••..•.•• :,.: . . . . . • . . . • . . . • . . : . . . • . .•.•..• • ,.: ' ...•... : . • . . . . . • . . . . ' . • . . . . ,:".: ".:.<: ;.:,:,::,.:- .... <.'.- .' ... <> ... : . . . : : . & : .. ' .",:"/'< Shakewell--anddiscaid200ul of·thissolution.· .•••. ; .•. , " ..•.• • A.· .•.•.•.•. : . • • • .•. : .•. ~ •. : · ..•.. .•..••••.....•.........•. : .•... ~ , ' " . ·.•...•.•. : ".f),', immunog19buhnes ·to ••.•... ..Add.·.· ~<ulpeiQltid~~ei~Qili~fi:atedFa.h"'H ....•.......•............. i·(19):8rarl:~::~o~r11!::/r~:~;;;1Infibod{e~an~(addsecoidar~·.·· . ' . ' . . . . . , . . . • . . . . . . . • . . . . . . • . . . . . . . ···antiboMes for· 30miiitttes:> . oJ- s h g d i a i n i n o s t r n t r e t u (20) Wash withalbuminized.PBS and PBS.
' ¥ " rabb~~oglobulins. h " " i ; , , ; " ' " ~O " " " i ~ ~ . , ) , ~ : ~::.:'; '.~ CIHRT Exhibit P-2156 Page 3 Eastern Health - source: Terry Gulliver Page 259 Volume 1 4 ····· .. ········· ." ' r . ,", 3 - - , . Teritary Antibody r secondary antibody li~j..ng~P;;~xidase di 1 utions as ) Prepare ( 2 1 ' unoglobulins t o conjugated sWin~ /"' / s : m i n u t e as before an~nfibOdY f Q l f (22) Prepare / - e s . ' . . , * ( 2 3 ) diilim, f>fPBS. ., n g h PBS and ret-urn t o s t a i n i r l w i t W a s h j e - : ,Revelation of Peroxidase S t a i n i n g - . " . (24)· PtepareDAB: . ; forfislidesinl0· {diaminobenzidine} A I l e 6 m g o f n . D i s s o l v . . " . " ' .·';;'{;ij )l;~"'1i'" tl!v.ealtoo. o r 3 to ·5minute§..,-"A,DoMt.att~tto· slide f . :.. . .. maxim1llli 15 sl:i.de's}.' manysiidesat onetirne~ . " : " . - , . ' p H20 to stop reiletiOll, t h .old t a 6 ) R i n s e w i ( 2 ': .. ',"-' . . y .... ?' ... < .. : . . .... ).:. ·~1tlif~t()astainitigjatof"atei. . , • . • . ) ... : . . , > : . : . . . _.<.: \ . , ' . : .. :.>-~.: -:- .. ", - . " . . -' :.: ......... :.,~.:. ... :'::-: '.: - .. ' . . . : . _ : : . ' . " " , ' . (33) Xyleno;. . h Permount t w i t M o i l n
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