TripleStain IHC Kit: R&R&M on rodent tissue (DAB , AP/Red & HRP/Green) (ab183295)

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Overview

  • Product name

    TripleStain IHC Kit: R&R&M on rodent tissue (DAB , AP/Red & HRP/Green)

  • Assay type

    Polymer Multiplex

  • Product overview

    Abcam's TripleStain IHC Kit (ab183295) is designed to be uses with user-supplied one mouse and two rabbit primary antibodies to detect three distinct antigens on a single mouse/rat tissue or cell sample. This kit has been tested on tissue specimens that are paraffin embedded; however it may also be used on frozen or freshly prepared monolayer cell smears.

     

    Triple staining uses traditional methods in immunohistostaining to reveal three distinct antigens and their co-expression on a single tissue. Abcam's TripleStain IHC Kit (ab183295) supplies polymer enzyme conjugates: Mouse HRP Polymer, Rabbit HRP Polymer and Rabbit AP Polymer with three substrates/chromogens; DAB (brown), Emerald (green), and Permanent Red (Red). Abcam's TripleStain IHC Kit (ab183295) is a non-biotin system, avoiding non-specific binding caused by endogenous biotin. This kit has been optimized to have no cross detection when detecting two primary antibodies from the same host species using a unique blocking system. Simplified steps allow the user to complete triple staining within 5 hours (without antigen retrieval) or 6-7 hours (with antigen retrieval). The well tested protocol allows the user to permanently mount slides with coverslip.

  • Tested applications

    Suitable for: IHC-P, IHC-Frmore details

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components Identifier 240 ml 72 ml 24 ml
    Non-aqueous Mounting Medium —
    Antibody Blocker (40x) 1 x 100ml 1 x 50ml 2 x 15ml
    Blocker A 1 x 120ml 2 x 18ml 1 x 12ml
    Blocker B 1 x 120ml 2 x 18ml 1 x 12ml
    DAB Chromogen (20x) 1 x 6ml 1 x 2ml 1 x 1.5ml
    DAB Substrate 1 x 120ml 2 x 18ml 1 x 15ml
    Emerald Chromogen 1 x 120ml 2 x 18ml 1 x 15ml
    Mouse HRP Polymer 1 x 60ml 1 x 18ml 1 x 6ml
    Mouse Primer 1 x 120ml 1 x 36ml 1 x 12ml
    4 x 9ml 1 x 12ml
    Permanent Red Activator (5x) 2 x 12ml 1 x 7.2ml 1 x 3ml
    Permanent Red Chromogen (100x) 1 x 1.2ml 1 x 360µl 1 x 150µl
    Permanent Red Substrate 1 x 120ml 2 x 18ml 1 x 15ml
    Plastic pipette 1 unit 1 unit 1 unit
    Rabbit AP Polymer 1 x 60ml 1 x 18ml 1 x 6ml
    Rabbit HRP Polymer 1 x 120ml 2 x 18ml 1 x 12ml

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab183295 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.

Protocols

References

ab183295 has not yet been referenced specifically in any publications.

Publishing research using ab183295? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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Question


1. Can cells block with 3% H2O2 and levamisole

2. The step 11 prepare DAB working solution but not mentioned any use, only store and cab be used within 7 hours. But in step 15 prepared Permanent Red working solution and used immediately in next step.

3. In step 12 apply the polymer mixture again, why? In step 7 prepared polymer mixture and applied in steps 8 &9. In step 11, not do DAB staining why in step 12 apply polymer mixture again?

4. step 19 To block the previous used antibodies: should do the same thing for cells?

5. In steps 25 and 28 applied Blocker A and B for what, to block what? 3% H2O2 to block endogenous background, blocker A and B for what?

6. Emerald Chromogen including what context and use for what?



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Answer

Here are answers to your questions.

For the TripleStain IHC kit I have some questions about the protocol attached
Q1. Can cells block with 3% H2O2 and levamisole
A1. Yes, 3% H2O2 and levamisole can be used to block endogenous HRP and AP for cells.

Q2. The step 11 prepare DAB working solution but not mentioned any use, only store and cab be used within 7 hours. But in step 15 prepared Permanent Red working solution and used immediately in next step. Is there a time limit to when this can be used?
A2. Make enough DAB mix by adding 1 drop of Reagent 4B (DAB Chromogen) in 1mL of Reagent 4A (DAB Substrate). Mix well. Use within 7 hours store at 4℃.
Apply 1 to 2 drops (50-100µL) of your DAB mixture to cover the tissue completely.
Incubate for 5min.
Rinse thoroughly with distilled water.

Wash with 1xTBS-T 3 times for 2 minutes each.
At step7, it always requires researcher applying fresh made permanent red working solution on tissue. Sensitivity will decrease if they do not use fresh made permanent red.
Q3. In step 12 apply the polymer mixture again, why? In step 7 prepared polymer mixture and applied in steps 8 &9. In step 11, not do DAB staining why in step 12 apply polymer mixture again?

A3. In step 12 the DAB chromagen solution is applied, not the antibody mixture.

Q4. step 19 To block the previous used antibodies: should do the same thing for cells?

A4. Yes.

Q5. In steps 25 and 28 applied Blocker A and B for what, to block what? 3% H2O2 to block endogenous background, blocker A and B for what?

A5. Primary antibodies already added to tissue must be blocked to prevent Rabbit HRP Polymer (added in step 34) from binding to them

Q6. Emerald Chromogen including what context and use for what?

A6. Emerald green is the AP chromagen.

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