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參數(shù)資料
| 型號(hào)： | A701 |
| 廠商： | Electronic Theatre Controls, Inc. |
| 英文描述： | A701: Test slide, various cancers plus corresponding normal |
| 中文描述： | A701：測(cè)試幻燈片，各種癌癥加上相應(yīng)的正常 |
| 文件頁(yè)數(shù)： | 9/10頁(yè) |
| 文件大?。?/td> | 217K |
| 代理商： | A701 |
Appendix: application protocol 2
Immunohistochemistry
For research use only
IHC(immunohistochemistry)
Immunohistochemistry is an exquisitely sensitive method for locating an antigen within a cell or tissue through a
high-resolution image (a single cell among thousands or millions). The method is based on the use of a primary
antibody binding specifically to its cognate antigen. The bound antibody is then visualized by colorimetric or
fluorescent detection methods.
IHC procedure
The protocol needs to be optimized for antibody you
may want to test and/or you might need to follow
instructions from suppliers.
Antigen retrieval method
During the preparation of tissues for staining,
antigens are heavily modified by the fixatives
frequently on free amino acid groups. Because they
can be hidden by other molecules, antigen retrieval
procedure is required to counter these changes.
There are several methods for antigen retrieval. The
selection is made according to the experimental
purposes. If the experiment is the conditioning
process with first trial of that antibody, various
methods need to be tried.
①
Dry a slide at 58
℃
overnight
②
Deparaffinize in xylene
③
Hydrate the slide in gradient ethanol
④
Retrieve antigen (see left section)
⑤
Dip in 3% H
2
O
2
10-15 min and washing buffer
3 X 5 min
⑥
Block with normal serum
1. Direct method
⑦
Biotin-tagged Primary Ab for 1~2 hr at RT or
37
℃
incubator or for overnight at 4
℃
(don’t
wash, just change the blocking solution for
primary antibody) and washing buffer 2 X 5 min
2. Indirect method
⑦
-1. Biotin-tagged primary Ab for 1~2 hr at RT or
37
℃
incubator or for overnight at 4
℃
(don’t
wash, just change the blocking solution for
primary antibody) and washing buffer 2 X 5 min
⑦
-2. Biotin-tagged secondary Ab for 10-15min at
RT and washing buffer 2 X 5 min
⑧
ABC reagent (streptavidin-HRP) for 10~15min
and washing buffer for 2~3 X 5 min
⑨
Fresh chromogen (DAB or AEC) for 1~3 min and
Wash with tap water
⑩
Counter stain (the nucleus) with Hematoxylin
or methyl green
-1. When Hematoxylin is used :
dehydrate in gradient ethanol (70% to 100%)
and clear with xylene,
mount with insoluble mounting medium
(eg. balsam)
-2. When methyl green is used : just dry and mount
with soluble mounting medium (eg. glycerin or
gelatin)
-3. When AEC is used for chromogen :
just dry and mount with soluble mounting medium
1. Proteolytic enzyme pre-treatment method
Cleave the bonds formed from the fixation process.
Enzymes routinely used include
pepsin.The concentration and reaction time must be
controlled since the excess enzyme treatment can
damage the target antigens.
trypsin
,
,
pronase
or
- pronase: 0.05%(W/V) in PBS or
- trypsin : 0.05%(V/V) in PBS
- pepsin : 0.05%(V/V) in 2N HCl
①
Incubate in one of the above solutions
at RT or 37
℃
for 18 min
②
Dip in cold DW
2. Heat-induced antigen retrieval method
Antigens fixed in formalin are hidden by fixative and
calcium ions. Chelating or precipitating these
calcium ions by specific solutions like citrate buffer,
EDTA and EGTA with heating can cleave these
bonds.
①
Place the slides into a rack
②
Immerse the slides in citrate buffer*
③
Move the entire container into microwave oven
④
Microwave the slides at maximum watt for 4 X 5 min
( after each cycle, replenish any lost liquid from
the slide container by addition of DW)
⑤
Remove the container and allow it to cool to RT
⑥
Wash with appropriate washing buffer
*citrate buffer : 0.01M citric acid, pH 6.0
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