Ncl-rtu-d

Sir I want to know that what is the Staining procedure for the NCL-RTU-D so can you please tell me the same

[Kiran Chandar]

Hey the The RTU Novostain Universal Detection Kit contains three reagents in ready-to-use form:

(1) prediluted normal horse serum,

(2) predilutedbiotinylated secondary antibody which recognizes rabbit IgG, mouse IgG and mouse IgM and

(3) a prediluted streptavidin peroxidaseconjugate. Each is supplied in convenient dropper bottles.


Staining Procedure for Paraffin Sections
1.
Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohols. Determine if any unmasking
pretreatment(s) is required for specific primary antibodies by referring to the manufacturer
’s data sheet.
2.
Rinse briefly in tap water then in PBS (if endogenous peroxidase is present, incubate solutions for 10 minutes in 0.5% v/v H2O2
in methanol or 3% v/v H2O2
in tap water for 5 minutes).
3.
Incubate sections at 25 oC for about 10 minutes in prediluted blocking serum.
4.
Blot excess serum from sections.
5.
Incubate sections at 25 oC either in primary antibody diluted in buffer containing 5% blocking serum (NCL-H-SERUM) or Ready to Use Primary Antibody.
6.
Wash slides for 5 minutes in PBS.
7.
Incubate sections at 25 oC in prediluted biotinylated universal secondary antibody for 10 minutes.
8.
Wash sections for 5 minutes with PBS.
9.
Incubate sections at 25 oC in prediluted streptavidin/peroxidase complex reagent for 10 minutes.
10.
Wash sections for 5 minutes with PBS
11.
Incubate sections in peroxidase substrate solution until desired stain intensity develops (see note 2 for approximate development
times).
12.
Rinse sections in tap water.
13.
Counterstain, clear and mount.
* The length of incubation times vary depending on the concentration of primary antibody. Generally, primary antibody concentrations
should be such that optimal staining is achieved with incubation times of 15 minutes to 1 hour.
In most cases, washes between reagent incubations can be shortened to brief rinses.
Staining Procedure for Frozen Sections
This procedure is generally appropriate for frozen sections, cell smears or cytospin preparations.
1.
Sections should be either unfixed, acetone-fixed or appropriate fixative for the antigen in question.
2.
If quenching of endogenous peroxidase is required, use gentle H2O2
blocking to reduce the risk of antigen destruction or tissue
loss: 0.3% H2O2
in 0.3% NHS in PBS for 5 minutes; or 0.3% H2O2
in methanol for 30 minutes, or use other published methods (eg
Andrew S M and Jasani B. Histochemistry Journal. 19: 426–430 (1987)). If necessary, H2O2
treatment may also be performed after
the biotinylated secondary antibody step.
3.
Follow steps 2–13 of the staining procedure recommended for paraffin sections.
Notes
1.
Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in pre