Immunohistochemistry kit

【資料簡(jiǎn)介】

Immunohistochemistry kit     
The  kit   is  based  on   the  HRP   streptavidin  Conjugate  (HRP   streptavidin 
Conjugate of HRP SA). It can be used to detect specific Thy1 antigen in the 
Blood,  cell or organization. The kit has high sensitivity and specificity, accurate 
qualitative and quantitative analysis, and a clear background. First of all, after 
the combination of primary antibody and the corresponding target antigen of 
the Thy1 used before, combine the biochemical secondary antibody    with the 
primary antibody with an anti specific way, finally join the HRP SA, then the 
antigen   specific primary antibody   biotinylated secondary antibody HRP SA 
complex is formed, and you can observe the imaging with a microscope. 
Principles and main steps of the kit 
The reagent in the kit       
Reagent A   Transparent liquid    0.1% Triton X 100   10 mL (optional)
Reagent B   Blocking buffer (closed) 
20 mL
Reagent  C      (Original import packing) diluted ready to Thy1 antibody (2.5ml)
Reagent  D      (Original imported packing) biotinylated goat anti rabbit IgG(1 
branch) 
Reagent  E     (Concentration of 1.5 mg / mL, the dilution ratio is 1:300 ~ 1:500) 
50   L + antibody diluent 20ml  
Reagent  F     HRP SA complex 1 branch(Concentration of 1   M, dilution ratio 
1:50 ~ 1:200) 100   L
 Own reagents of the user        
1. 10mM TBS（pH7.2~7.4）
Three hydroxy amino   methane 1.21g 
Sodium chloride 7.6g 
Add distilled water 800ml,concentrated hydrochloric acid,adjust pH to 7.2 to 
7.4 
TBS T：TBS+Tween 20（0.05% volume ratio）
2. Antigen retrieval buffers(By antigen detection and repair fluid) 
Citrate buffer 10mM pH 6.0 r 
Citric acid 0.38g 
Citrate Sodium 2.45g 
Add distilled water 800ml, concentrated hydrochloric acid,adjust pH to 6.0,
the final volume to 1000mL Or 0.5M EDTA repair solution 
EDTA?2H2O 186.1g 
Add distilled water 700ml, 10mM of NaOH ,adjust pH to 8.0,the final volume 
to 1000ml 
3.  10 mL of buffered glycerol mounting medium 
4. Tween 205 mL
Immunohistochemical staining of paraffin-embedded tissue     
Experimental procedure (proposed): 
Paraffin embedded tissue sections of 4  m thickness 
1.Drilled  piece:  Place  the  slice  on  the  sliced  rack, bake  at  60℃  constant 
temperature oven for at least 1hour; 
2. Dewaxing: Put the slice into a container filled with xylene dewaxing for three 
times; 
3.Hydration: Hydrate the  slice through the downstream  alcohol, pure ethanol 
5min, ethanol of 95% concentration for 2 times(2 min per time) ethanol of 85% 
concentration  for  2 min,  ethanol  of  75%  concentration  for  2  min,  flush  with 
water, ddH2O 2×2min；
4.Antigen  retrieval:  Retrieve   the  antigen   according  to  the   recommended 
method  of  antibody   instructions,  usually  using  high   pressure,  microwave 
(temperature of 98 to 100 ° C) or enzymatic digestion repair method, 
natural cooling，tap water，ddH2O washing 2 × 2min, TBS washing (2 × 2min) 
(Note: some antigen Needless to repair directly to step 5 closing) ；
5. Closing: dropping reagent B, incubating in wet boxes (37 °C) for 30 min；
6.Add primary antibody: dropping reagent C, incubating in wet boxes (37 °C) 
for 2 hour；
7.Washing：  TBS T washing（3×5 min）；
8.Closing：  dropping reagent B，incubating in wet boxes (37 °C) for 10min；
9.Add secondary antibody:  dropping  the biotinylated secondary  antibody(reagent D)
incubating in wet boxes (37 °C) for 30min；
10.Washing：  TBS T washing（3×5 min）；
11.Closing：  Dropping reagent Tween 20，incubating in wet boxes (37 °C) for 
20min；
12．Add HRP SA：dropping reagent E which is diluted with reagent C  （1：
50~200，final concentration 5~20 nM）,incubating in wet boxes (37 °C) for 
30min；
13.Washing：  TBS T washing（3×5 min）, TBS washing（2×5 min）；
14. Color: DAB solution (reagent F) color ；
15.Stained:Tap the full flush, recolor, transparent；
16.  Closing  the  slice: When  the  tissue  sample  is dry,  closing  the  slice  with 
reagent buffered glycerol mounting medium；
17. Observing the imaging: Observe the imaging with microscope； 
 Notes :   
1.  After it is retrieved, it should be cooled with natural way, it must be rinsed 
before removing the slice.  Quench may result in crystallization or antigen 
closed. 
2.  The buffer must ensure that the slices can be soaked , the citrate buffer 
used before can not be used repeatedly. 
3.  If the reagent is trace concentrate, the inner lid and wall attached to the