r . ,", 3 - - , . Teritary Antibody r secondary antibody - - PDF document

SLIDE 1

E a s t e r n H e a l t h

• s
• u

r c e : T e r r y G u l l i v e r

Day I

Prepare S l i d e s : Cut sections @ 3 microns on clear glass s H d e .

• ' Place

i n

58-60 degree C. incubator overnight '

,Day

I I

• Place 200 mIl/H) PBS

i n

a 37 degree c.in~tl!l)lf.

• W

e i g h

• u

t . 2 . g m hyps~n.

• Make

u p

albumini~edl/lO

P B S :

V

• l

u m e 14 P a g e 2 5 7

CIHRT Exhibit P-2156 Page 1

SLIDE 2

E a s t e r n H e a l t h

• s
• u

r c e : T e r r y G u l l i v e r

.:<'.

V

• l

u m e 14 P a g e 2 5 8 ( 8 )

(9)

• Abs. alcohol ...

Chloroform

• 2
• "

,

• '

"

/'

5 min.

5

m i r i .

" j

5

min;

5 m in.

miliimum

(13)

(10) Acetone

/5 min.

/

/ /

(11) Cold

a p

H 2

........

, ~ ....

///

(12)

~1'

uminized

P B S /

. Primary

A n t i b

• d

Y '

. Prepare dilutions of primarYantib()dYUSi.ngaIhl!llJliWi?l~>~\·· 1 4 0 - 200' ul per

s l i d e .

>(14)' Prepare.slides>ollebycine by . drYing()~t~ideandleavingi

: : ;

tilrnof .the ,alhuiiliniji,ed

· . P B s . a r

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n d :lhe>sectioll

(ROArK

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<BUBBLE9)':(110#ci'1'I.ETT!l~

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e r 5 ~ ~ t i .

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i<..•..... .

time

) f

• r30IJlinlltes/i~a.huntidificaJiQii

¢~~~er.

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...•. . · · · · ( 1 6 ) . Wash

each'

slide~~z:efll.lIywhh iIlopas{aibtinu.nize~)using.

asqueezeholUe;" .'

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.

. :.

.

. . .' . (l7)'l'ransfer t

• a staining

d i s h

• f the albilrninill'.edPBS ..

.;)~ecandarY Antibody . • "

.·~X(18),preparedi luti~n~stpiows:

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'lor20~licl~{/<i>

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Shakewell--anddiscaid200ul of·thissolution.·

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immunog19buhnes ·to

....•.......•............. i·(19):8rarl:~::~o~r11!::/r~:~;;;1Infibod{e~an~(addsecoidar~·.··

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. (20) Wash withalbuminized.PBS and r e t u r n

t

• s

t a i n i n g

d i s h

• J-

PBS.

CIHRT Exhibit P-2156 Page 2

SLIDE 3

Eastern Health - source: Terry Gulliver

r

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. ,",

.

,

• 3
• Volume

1 4 Page 259

Teritary Antibody

' ( 2 1 ) Prepare

di 1utions as

r secondary antibody li~j..ng~P;;~xidase conjugated sWin~ unoglobulins

t

• rabb~~oglobulins.

/

/"'

. ' . .

(22) Prepare

/

• e

s

as before an~nfibOdY

f Q l f

~O

m i n u t e s :

, * ( 2 3 )

W a s h j e

• r

l w i t h PBS and ret-urn t

• s

t a i n i n g

diilim, f>fPBS.

.,

,Revelation of Peroxidase S t a i n i n g :

'.~

• .

"

. . (24)· PtepareDAB:

. D i s s

• l

v e 6

m g

• f

n A I l

{diaminobenzidine}

forfislidesinl0·

;

.·';;'{;ij)l;~"'1i'"

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" ' "

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slide f

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3 to ·5minute§..,-"A,DoMt.att~tto· tl!v.ealtoo.

manysiidesat onetirne~

maxim1llli 15 sl:i.de's}.' ..

.

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.

• (

2 6 ) R i n s e

w i t h .old t a p H20 to stop reiletiOll,

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l n t w i t h Permount

CIHRT Exhibit P-2156 Page 3