UniversalTM One-step Immunohistochemistry (IHC) Kit (For R&D Use only)

UniversalTM One-step Immunohistochemistry (IHC) Kit
Product Name & Catalog Number:
Product Name Catalog # Size Content

UniversalTM One-step IHC Kit-R

With DAB Substrate

(Suitable for rabbit primary antibodies) 
IHC-R60 1 kit
(For 200 slides)
 
IHC Pretreatment Solution (40ML),
IHC Enhancer (20ML),
10X IHC Probe-R0 (2ML),
10X Rapid Wash Solution (50ML),
100X DAB Solution Set (1ML each)

UniversalTM One-step IHC Kit-M With DAB Substrate

(Suitable for mouse primary antibodies) 
IHC-M60 1 kit
(For 200 slides)
IHC Pretreatment Solution (40ML),
IHC Enhancer (20ML),
10X IHC Probe-M0 (2ML),
10X Rapid Wash Solution (50ML),
100X DAB Solution Set (1ML each)

UniversalTM One-step IHC Kit-G With DAB Substrate

(Suitable for goat primary antibodies) 
IHC-G60 1 kit
(For 200 slides)
 
IHC Pretreatment Solution (40 ML),
IHC Enhancer (20 ML),
10X IHC Probe-G0 (2ML),
10X Rapid Wash Solution (50 ML),
100X DAB Solution Set (1ML each)

UniversalTM One-step IHC Kit-R With BCIP/NBT Substrate

(Suitable for rabbit primary antibodies)
IHC-R61 1 kit
(For 200 slides)
IHC Pretreatment Solution (40ML),
IHC Enhancer (20ML),
10X IHC Probe-R1 (2ML),
10X Rapid Wash Solution (50ML),
BCIP/NBT Substrate (20ML)

UniversalTM One-step IHC Kit-M With BCIP/NBT Substrate

(Suitable for mouse primary antibodies) 
IHC-M61 1 kit
(For 200 slides)
IHC Pretreatment Solution (40ML),
IHC Enhancer (20ML),
10X IHC Probe-M1 (2ML),
10X Rapid Wash Solution (50ML),
BCIP/NBT Substrate (20ML)

UniversalTM One-step IHC Kit-G With BCIP/NBT Substrate

(Suitable for goat primary antibodies) 
IHC-G61 1 kit
(For 200 slides)
IHC Pretreatment Solution (40 ML),
IHC Enhancer (20 ML),
10X IHC Probe-G1 (2ML),
10X Rapid Wash Solution (50 ML),
BCIP/NBT Substrate (20ML)
Product Description:

UniversalTM One-step IHC is a latest breakthrough technology (patent pending) for performing Immunohistochemistry (IHC) or Immunocytochemistry (ICC). Unlike regular (classical) IHC/ICC, the One-step IHC/ICC technology combines blocking, primary antibody staining, washing and secondary antibody staining into a rapid one-step reaction. Therefore, using UniversalTM One-step IHC kit, your IHC/ICC becomes so simple and is no longer a time-consuming and labor-intensive procedure. The One-step IHC kit is a universal kit, which is suitable for most primary antibodies and no need of secondary antibody. Currently, we provide six types of UniversalTM One-step IHC kits as shown on the above table. Each kit is sufficient to stain 200 slides.

Application:

Immunohistochemistry (IHC) or Immunocytochemistry (ICC).

Features:

Easy, a simple One-step reaction instead of the 4-step procedure: blocking, primary antibody-binding, washing and 2nd antibody-binding.

Rapid, just 1~2 hrs instead of 4~5 hrs.

Universal, suitable for most primary antibodies and no need of secondary antibody.

Sensitive, comparable sensitivity with regular IHC or ICC.

Reproducible, highly reproducible results.

Storage: Pretreatment Solution and Enhancer: for continuous use, store in 2~8 <C for up to 1 month, or store in -20 <C for up to a year.

IHC Probe and Substrate: for continuous use, store in -20 <C for up to 6 months or store in -70 <C manual defrost freezer for up to a year. Repeated freezing and thawing is not recommended.

Rapid Wash Solution: store in room temperature for up to one year.

Note: Kit will be shipped in ambient temperature.

Comparison of One-step IHC with Regular IHC:

Protocol:

I. Other required materials: Primary antibody: Purified monoclonal or affinity-purified polyclonal antibodies (antigen-specific IgGs) are preferred. Although unpurified antibody or serum can also be used for the One-step IHC, user may need to optimize staining conditions to avoid non-specific binding.

Note: We provide different type of UniversalTM One-step IHC kits. Kits labeled with CR, -M and CG are suitable for Rabbit, Mouse and Goat primary antibodies, respectively. . Fixatives: 30% Buffer-neutralized Formalin or Z-fix. Counter staining Reagent: Mayer¨s hematoxylin or other proper counter staining reagents.

II. Reagent preparation: One-step IHC Antibody Mixture: For 10 slides, mix 5-10ul of primary antibody and 100ul IHC Probe (supplied in the kit) with 1ml of IHC Enhancer (supplied in the kit). The amount of primary antibody may vary depending on the quality of the antibody and it may need optimization by user to obtain best results. 1X Rapid Wash Solution: Dilute whole bottle of 10X Rapid Wash Solution (supplied in the kit) to 10 time volume with distilled water and store in room temperature. 1X DAB Mixture (Make this mixture just before use): For 10 slides, mix 20~40ul of 100X DAB solution (supplied in the kit), 20~40ul of 100X D-buffer (supplied in the kit) with 2 ml of distilled water.

III. Procedure: 1. Fix: Incubate sample (tissue or cells) on slide with Formalin (or other proper fixatives) for 20 min and then rinse the slide with 600ul PBS. Note: this step is same to the method for regular IHC or ICC.

2. Pretreatment: Incubate the pre-fixed tissue section or cells on slide with 100ul (per slide) of Pretreatment Solution for 10 minutes at room temperature in order to inactivate the endogenous peroxidase, and then rinse this slide once with 600ul PBS.

3. One-step Reaction: Incubate the above slide (from Step 2) with 100ul of the pre-prepared One-step IHC Antibody Mixture (a mixture of primary antibody, IHC enhancer and IHC Probe) for 30~60 minute at room temperature.

4. Wash: Rinse the slide three times with 600ul of 1X Rapid Wash Solution and once with 2ml of PBS.

5. Development: Apply 200ul of the pre-prepared 1XDAB mixture (for kits IHC-R60, IHC-M60and IHC-G60) or BCIP/NBT substrate mixture (for kits IHC-R61, IHC-M61 and IHC-G61) to the washed section on slide and incubate for an appropriate time (approximate 2-20 min) until a desired color appears. To stop the development, simply rinse slide with distilled water.

6. Counter-staining: Incubate the slide with the solution of Mayer¨s hermatoxyin for 0.5-5 min at room temperature until a desired background color appears. Rinse the slide with distilled water. Note: this step is same to the method for regular IHC or ICC.

7. After Staining: Take photos and/or mount the section with glycerol gelatin and a sealing cover-slip with clear nail polish. Note: This step must be performed according to the standard procedure used for regular IHC or ICC.

Example: Comparison of One-step ICC with Regular ICC in Detection of the Expression of a Flag-tagged protein in A549 Cells. A549 cells cultured in 96-well plate were directly fixed on the wells of tissue culture plate and then pretreated with the kit-provided pretreatment solution. Expressed Flag-tagged proteins in cells were detected using mouse anti-Flag antibody by a regular ICC procedure (A) or by a one-step IHC procedure (B) or by a one-step IHC procedure but without using primary antibody (C). As showed in the picture, the results by both methods are very similar but the testing time and procedure are greatly different. A: 5 hours (using regular method). B: <1 hour (using One-step IHC kit). C: One-step ICC without primary antibody (as a negative control). Note: the brown color signals indicated the positive stains using anti-Flag antibody and DAB substrate.

Troubleshooting:
Problem Probable Cause Solution
Weak Signal or No Staining Inadequate deparaffinization Deparaffinize sections longer or change fresh xylene
Inactive primary antibodies Replace with a new batch of antibodies
Antibodies do not work due to improper storage Aliquot antibodies into smaller volumes and store in freezer (-20 to -70 ≧) and avoid repeated freeze and thaw cycles. Or store antibodies according to manufacture's instructions.
Concentration of primary antibody or IHC probe was too low Increase the concentration of primary and/or One-step IHC probe. Or run a serial dilution test to determine the optimal dilution that gives the best signal to noise ratio
Incorrect primary antibody type Check the label of IHC Kit and select compatible primary antibodies and One-Step IHC Kit. Kits labeled with -R are suitable for rabbit primary antibodies, with -M suitable for mouse primary antibodies, and with -G suitable for goat primary antibodies.
Poor specificity of primary antibodies. Use purified monoclonal or affinity-purified polyclonal primary antibodies.
Inadequate antibody incubation time Increase antibody incubation time
Inadequate or improper tissue fixation Increase duration of postfixation or try different fixatives
Tissue overfixation Reduce the duration of postfixation. If the tissue has already been overfixed, perform an appropriate or recommended antigen retrieval antigen retrieval procedure.
Defective One-step IHC probe Replace with a new batch of One-step IHC probe.
Inadequate substrate incubation time Increase the substrate incubation time.
Over-staining The concentration of primary and/or One-step IHC probe was too high Reduce the concentration of antibody or IHC probe, or perform a titration to determine the optimal dilution for primary and IHC probe
Incubation time was too long Reduce incubation time
Incubation temperature was too high Reduce incubation temperature
Substrate incubation time was too long Reduce substrate incubation time
Sections dried out Avoid sections being dried out
Inadequate washing of sections Wash at least 3 times between steps
High Background Tissue contains endogenous peroxidase Increase incubation with pre-treatment solution or 3% hydrogen peroxide to block endogenous enzyme activities prior to incubation of antibody.
Non-specific binding of primary antibodies to tissue or antibody concentration was too high Non-specific binding may be reduced by using higher dilution of primary antibodies or by using purified monoclonal or affinity-purified polyclonal primary antibodies.
Diffusion of tissue antigen due to inadequate fixation Increase duration of postfixation
Sections dried out Avoid sections being dried out