PROTOCOL- Immunocytochemistry

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IMMUNOCYTOCHEMICAL IDENTIFICATION OF PROLIFERATING CELLS IN VASCULAR TISSUE (ANTI-BrdU); Diaminobenzadine (DAB) brown detection0 U4 Z+ R$ A- A7 Y

6 E4 O+ L4 `3 [4 L* |, ^2 f( g1 Sections of paraformaldehyde fixed, OCT-embedded vascular tissue are sectioned at 7 to 10 mm and stored, desiccated at -70°C until needed. - _! l) l% @! O0 X4 `$ A
2 Thaw slides immediately before use, usually 10-30 min. prior to beginning the protocol. " H: H6 H* V% w  I
3 Fix in acetone 5 min., air dry for approximately 20 min., RT. 7 P; o7 G* a1 W, G
4 Immerse in 0.3% H2O2/Methanol for 15 min., RT. (450µl H2O2 in 150ml Methanol).
4 c+ }' q4 E5 t! e5 Immerse in Proteinase K/ 1xPBS for 10 min., RT (7.5µl Proteinase K of 20 mg/ml stock in 150ml 1x PBS for a final concentration of 1µg/ml). / L& w3 e' Z! O0 O. w
6 Rehydrate in 1x PBS twice for 5 min. each, RT. ; y& \. t! s" J1 ?9 ?: O/ g
7 Immerse in 4N HCl for 10 min., RT.
/ ~( y+ l- M% l+ ]7 v8 Immerse in 1x TBE, pH 8.4 for 5 min., RT. Check pH of buffer with pH paper to see if it is neutral. ! G2 R' Y; w) [' _9 I& P% t4 W2 ^
9 Immerse in 1x PBS. Check pH of buffer after 2 min. Apply primary antibody when pH is 7.0-7.5.   F) n$ p& ?' m/ w
10 Prepare the working dilution of the primary antibody in 1.0% crystalline-grade Bovine Serum Album (BSA) in 1x PBS. Use BrdU antibody (Dako Catalog No. M744) at 1:20 dilution. Blot off PBS and apply 150µl of working dilution. Incubate sections in a humid chamber for 1 hour, RT. Blot off antibody, wash 2 times 5 min. each in 1x PBS.
$ N/ I! S7 W) d7 H1 ^11 Prepare the working dilution of the secondary antibody (Horse anti-mouse IgG-Vector Catalog No. BA2001) in 1.0% BSA/PBS, 2.0% normal horse serum. Prepare the secondary antibody at a 1/400 dilution. Apply 150µl and incubate 30 min., RT in a humid chamber. Blot off the antibody, wash 2 times 5 min. each in 1x PBS. . R# ~, T9 J' {; c' c) M: M& F8 n
12 Prepare the working dilution of ABC-Peroxidase complex or the Vector ABC-peroxidase Elite (Catalog No. PK-6100) before finishing the previous step. Mix 5ml 1x PBS, two drops of Solution A, and two drops of Solution B, and allow to sit at 4°C for 30 min. prior to use. Apply enough to cover the sections on the slide, and incubate for 1 hour. Blot off the solution, wash 2 times 5 min. each in 1x PBS, followed by 1 wash in 100mM Tris pH 8.2 for 5 min.
. V' K( v7 N. _/ R6 S/ o/ z13 Make up substrate solution immediately before use. Add 5 ml of sterile water, two drops of buffer from Vector DAB substrate kit (Catalog No. SK-4100), four drops DAB, two drops H2O2 , and two drops of Nickel from kit, in 15ml tube (wrap the tube in foil to protect the substrate from light) . Incubate in the dark for 20-30 min., stop color reaction by blotting off substrate and rinsing in dH2O water for 5-10 min. Caution: DAB is highly carcinogenic, so handle with gloves and work on absorbent towels. Inactivate solutions by pouring into a beaker containing 3% KMnO4 and 2% Na2CO3 in water, and then dispose down sink. 2 E' O4 b. t1 C' F  V
14 Counterstain with hematoxylin, 1% acid-alcohol, Scott's solution, dehydrate through graded alcohols, then xylenes per protocol below, and coverslip for viewing. $ `% R  \$ g- c2 G' Y
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Solutions:& b3 v- W5 R$ W
Hydrogen peroxide : @: Q# }+ O9 g0 q3 g7 |! x
SIGMA Catalog No. H-1009 (5 ml) - j& u- W' w$ t! r" ^* @! F* U& q, D
Proteinase K
% Y; N; A( s' j% a2 c/ D. A+ f, IDissolve Proteinase K (Sigma Catalog No. P-4914) in dH2O for a concentration of 20mg/ml.
" ?* N5 t  a# D4 i- j- M6 z1% BSA/PBS 8 N" h; L! u/ [7 V' K
1g Fraction V BSA in 100ml 1x PBS. Mix in a beaker with low heat. Aliquot in 15ml tubes and store at -20°C.
+ [: u: _! E  T, `3 z/ M) JHematoxylin : e9 y( f0 q1 Z4 u* }* u0 Z. x1 f) @
75ml Gill's hematoxylin no.2 in 75ml dH2O. - j0 K6 x2 u# L" ]: G* i) l
1% Acid-alcohol
' ~0 q- [; E! H( ?; @0 d# D7 K# s8 P2ml HCl in 198ml of 70% ethanol. + _# f7 u, D2 h% _: l: R; v$ v/ u7 D
Scott's Solution 5 C2 ?6 w$ H# A7 I
NaHCO3, 2g
7 [! Z7 A) I9 z. V& _
0 Y& l, }0 T/ L. A" ~7 DMgSO4.7H2O, 20g; y3 z* Y9 k+ @$ e
+ i8 U1 P! e2 U; c: K, ]9 o. q; R5 b
dH2O, 1000ml ; i" f9 z6 u% y* z1 y" g  q+ P
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Counterstaining Protocol:) I( W: S  \7 N; u$ D: x9 k
Hematoxylin - 10 sec. 3 e! V4 y2 p7 `9 ?" d- H' b2 B1 X; g
rinse in water until water is clear
- B3 t+ T+ }- K; m! l, |% `Very quick dip in 1% acid-alcohol (< 1 sec.) % J# s& U+ ~, L: A" h
rinse in water 5 h2 Y( ~# Q9 F% U* G
Scott's solution - 20 sec. 4 _- y8 f  W+ P- @( B
rinse in water
2 o; m, C# Z* JDehydrate through graded alcohols (70%, 95%, 95%, 100%, 100%) " l; G' i# W; p. ]+ d7 B! j
Xylenes # J- z+ b8 r. o
Coverslip% H% R+ q+ V) R

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From:http://www.emory.edu/WILCOX/brduDAB.html

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Description
9 I- `6 l- k; D- AProcedures for cell plating, fixing, antibody incubation and mounting6 m7 B. \0 k" L( L3 |
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Procedure+ s0 X$ D6 b1 W2 m
A. Plating:
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) p5 j) Z1 ^$ D$ W. PTo sterilize glass coverslips, dip in ethanol and flame. / O  Z, S4 [% D8 c; h6 p
We use 22x22x1 mm3 coverslips and put them in 6-well plates. + z1 G0 [; n/ z* J% Z
Seed 100,000 cells per well overnight and fix the next day.
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B. Fixation: * T/ Y6 @0 y* K2 p
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Remove the media and rinse once with PBS. . ]/ L8 i" c$ c, E1 g
Remove the PBS and immediately add -20°C methanol. (Do not allow the cells to dry.)
. M0 l3 R- a, y' K' hPut the plate in a -20°C freezer for 5 min.
" b+ _! I6 i; S0 [. N2 O1 QRemove the methanol and add PHEM buffer. Fixed cells are kept at 4°C in PHEM.
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C. Antibody incubation: 8 k. f: J  U5 N* @/ j) k4 E
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Block with appropriate sera (2.5 to 5%) in PHEM buffer for 1 hr with gentle rocking.
0 \) i, Q# v/ {Add primary antibody to the blocking buffer and incubate for 1 hr with gentle rocking. 9 O- s0 \) q- q' o8 b. ]  T
Remove and wash 4 x 10 min with PHEM buffer. ) E" q9 c- Z# J8 [
Add secondary antibody in PHEM buffer with sera and incubate for 30 min with gentle rocking. 4 t& \4 G: y1 @/ N, ~! d" x
Remove and wash 4 x 10 min with PHEM buffer. " W2 k1 j; ?4 i$ h9 v. i  G, Y# H
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D. Mounting: + ~& N; Q9 F) j: n- q9 D& [

! x+ ]9 P4 c1 f$ O; |! Q( O, lPick up coverslip with forceps and drain away excess buffer (can gently aspirate if desired). $ H$ w+ v9 U% B( i6 i9 I8 B* r0 s# _: {7 |' _
Put ~20 &micro;l "antifade" on slide and gently lay coverslip on top. ) f5 |  b* D0 W4 S9 R! U
After removing excess antifade, either by blotting with Kimwipe or aspirating, seal with clear nail polish.
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KEEP IN THE DARK AT ALL TIMES.
. ^5 X* I! D- F9 R( f* X9 KStore in -20°C freezer.
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) V& r2 R7 ]* |3 _" Y/ r  iRecipes
% n/ `8 w2 Y0 y1 ?5 P0 ~6 ^PHEM buffer: 1 m8 L* r0 R  M' b. S" ^
25 mM HEPES / P5 X5 \  m; c( e
10 mM EGTA 8 y# L( m! h: z' \5 S
60 mM PIPES + F' F4 O8 v* Q5 f( T# F
2 mM MgCl2 ; L' p: H9 y- L5 V
pH = 6.9 3 Z5 I/ G7 K0 L) X6 _* X. T2 ]
(Add in this order.) 0 I( \+ y# J1 B3 |; }
Antifade: 1 ml
0 B7 J; o8 Q8 J2 B3 r5 f1 mg p-phenylene diamine hydrochloride 9 V2 G8 g7 \4 J1 J  d
Dissolve in 0.1 ml 10x PBS (20 min at RT) # u& U4 R2 Q- O; ?5 Q) f8 S4 J
Add 0.9 ml 100% glycerol * V5 ^: i8 _- n1 p% J
Keep covered at all times and no vortexing. 9 f: _( P' k6 T7 ~: _
If it turns brown, it’s no good.
( r7 N) A4 s: oAliquot and store at -70°C. . ]$ V% r& s$ u( K2 h7 ]# o" _
# k% V! \$ @, M& x- a% p
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From:http://www.scienceboard.net/reso ... &protocol_id=20

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IMMUNOCYTOCHEMICAL IDENTIFICATION OF PROLIFERATING CELLS IN VASCULAR TISSUE (ANTI-BrdU); Vector Red Detection  t3 r2 z& ]5 \9 |  ^0 S: i
Josiah N. Wilcox and Romesh R. Subramanian - Emory University - September 20, 1993
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* O$ P  D0 W1 R0 p& m1 Sections of paraformaldehyde fixed, OCT-embedded vascular tissue are sectioned at 7 to 10 mm and stored, desiccated at -70°C until needed.
7 q7 y# H- ?+ R  q3 |  w! V2 Thaw slides immediately before use, usually 10-30 min. prior to beginning the protocol.
8 M& F! m- o$ u7 A( f3 Fix in acetone 5 min., air dry for approximately 20 min., RT.
5 t2 H! G1 U4 W, @1 l4 Immerse in Proteinase K/ 1xPBS for 10 min., RT (7.5&micro;l Proteinase K of 20 mg/ml stock in 150ml 1x PBS for a final concentration of 1&micro;g/ml).
- s; D4 _/ q# c0 ], E3 g1 @* p5 Rehydrate in 1x PBS twice for 5 min. each, RT.
8 i, q6 F' Q8 D* B( a# M6 Immerse in 4N HCl for 10 min., RT. ) B( J1 `" `7 @9 m8 ]0 e+ ?& R, O* m
7 Immerse in 1x TBE, pH 8.4 for 5 min., RT. Check pH of buffer with pH paper to see if it is neutral.
2 ^. B9 D& ]* Y' ^& v  B8 Immerse in 1x PBS. Check pH of buffer after 2 min. Apply primary antibody when pH is 7.0-7.5.
4 p4 H4 ~: I& V% i# n8 E& o9 c$ X9 Prepare the working dilution of the primary antibody in 1.0% crystalline-grade Bovine Serum Album (BSA) in 1x PBS. Use BrdU antibody (Dako Catalog No. M744) at 1:20 dilution. Blot off PBS and apply 150&micro;l of working dilution. Incubate sections in a humid chamber for 1 hour, RT. Blot off antibody, wash 2 times 5 min. each in 1x PBS.
* m* c" a3 O) N: w6 I10 Prepare the working dilution of the secondary antibody (Horse anti-mouse IgG-Vector Catalog No. BA2001) in 1.0% BSA/PBS, 2.0% normal horse serum. Prepare the secondary antibody at a 1/400 dilution. Apply 150&micro;l and incubate 30 min., RT in a humid chamber. Blot off the antibody, wash 2 times 5 min. each in 1x PBS. # q* V0 k) C& d  _
11Prepare the working dilution of ABC-Vector Red complex from the Vector ABC kit (Catalog No. AK-5000) before finishing the previous step. Mix 5ml PBS, two drops of Solution A, and two drops of Solution B, and allow to sit at 4°C for 30 min. prior to use. Apply enough to cover the sections on the slide, and incubate for 1 hour. Blot off the solution, wash 2 times 5 min. each in 1x PBS, followed by 1 wash in 100mM Tris pH 8.2 for 5 min.
, R) J" _" |+ o12 Make up Vector Red substrate solution (Catalog No. SK-5100) immediately before use. Incubate in the dark for 20-30 min, stop color reaction by blotting off substrate and rinsing in dH2O for 5-10 min.
# E" s% ~! G' z+ t% e1 S3 S13 Counterstain with hematoxylin, 1% acid-alcohol, Scott's solution, dehydrate through graded alcohols, then xylenes per protocol below, and coverslip for viewing. / b% K1 `: \1 o7 k3 w

; }( Z% p. Z6 E) ~" a, q/ x5 K) BSolutions:
+ |! d& s4 @% D# IProteinase K # }, @9 Y4 o- I8 m- s0 O! z
Dissolve Proteinase K (Sigma Catalog No. P-4914) in dH2O for a concentration of 20mg/ml.
+ Q* t; ^2 e/ V4 B7 s# s1% BSA/PBS
) ~$ p, ~# o( `1g Fraction V BSA in 100ml 1x PBS. Mix in a beaker with low heat. Aliquot in 15ml tubes and store at -20°C. 3 k, z' v* C. `  y* l
Hematoxylin
" \0 s" V5 _- D% d# l75ml Gill's hematoxylin no.2 in 75ml dH2O.
% O. w& r. S5 r! l- P9 i: {7 b1% Acid-alcohol ! y% ^7 n, S, W1 Y* x& {9 Y
2ml HCl in 198ml of 70% ethanol.
( X1 Y/ O0 A, f0 R8 W% @5 k1 cScott's Solution
, W% n% b) H, ^6 NNaHCO3, 2g
5 ]* \. c# D! p4 Y* o4 r
  m2 }8 e7 V- m$ gMgSO4.7H2O, 20g9 ]* |& u7 F4 ?3 c1 R7 R
2 I+ G4 ]; P& C, h6 B; Q
dH2O, 1000ml . {* a- }/ g% D0 K0 |! m, i2 I( M

# g' Y) L, _# b5 g( M2 d3 CCounterstaining Protocol:
7 C, E# z9 D/ N% f# z' SHematoxylin - 10 sec.
! F! n0 p9 x" U  B  irinse in water until water is clear : P  |) Y3 L& d9 z* }. y
Very quick dip in 1% acid-alcohol (< 1 sec.) : b" U1 Y+ v4 C* c' C. Z7 S
rinse in water + t" @1 [0 n" D& t1 ?0 Y- l! F
Scott's solution - 20 sec.
6 `; I8 y* v/ X; n5 i. |) c* Vrinse in water * Z0 H+ A- P: H9 q
Dehydrate through graded alcohols (70%, 95%, 95%, 100%, 100%)   X9 H$ h1 o% R' ^8 X) ?
Xylenes ) w# r9 y7 P( Z) K
Coverslip

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Introduction to Immunocytochemistry- T+ h2 e! @: O& R

- I6 h: w* J( p' N) I3 f3 eImmunocytochemistry is the name given to methods that use antibodies to detect the location of proteins within cells. The antibodies bind specifically to the protein being investigated. With electron microscopes this method is used with electron opaque markers which bind to the antibodies and visualize their location within the cell. One useful electron opaque marker is colloidal gold.( \, E& q3 P3 E; L6 `% Y
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For electron microscopy it is important to have preparation methods that open the cells and allow access of antibodies to the proteins (or antigens) they bind to without destroying the normal cellular organization (or morphology).
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. j6 N& G* F4 y* ]% G% T+ fOver the last ten years, specimen preparation techniques, especially those for immunocytochemistry, have reached high levels of sophistication and allow cells to be opened, labeled with antibodies and colloidal gold and yet retain good morphology. Some of these methods are briefly described in our introduction to electron microscopy.$ t7 M, d* c3 G% R  a7 F8 t6 }
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At the moment, the very best way to gain access to the inside of cells is by cutting sections through them. In this way the morphology is retained but antibodies gain total accessibility to the cut face of the cell. To cut sections thin enough to be examined in the electron microscope (approximately 40 - 100 nm), the cells must be made hard, either by embedding in Aresin or by freezing them in a cryoprotectant. Both methods are used routinely in the Center for Cell Imaging at Yale. For antigens which are only present in small amounts in the cell the most sensitive method is usually to label sections of frozen material.