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G1215-200T Immunohistochemistry Kit(HRP-conjugated Goat Anti-Rabbit IgG (H+L))

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G1215-200T Immunohistochemistry Kit(HRP-conjugated Goat Anti-Rabbit IgG (H+L))

This Kit is a highly sensitive two-step Immunohistochemistry kit, which is suitable for the primary antibody derived from Rabbit. The kit can be used in Western blot, and IHC applications. The color reaction principle is based on DAB reaction. Firstly, primary antibody of Rabbit origin binds to the antigen in test species. Then secondary antibody, HRP-conjugated Goat Anti-Rabbit IgG (H+L) combined with primary antibody. Next, DAB interacts with HRP, because DAB is the substrate of horseradish peroxidase (HRP). Under the catalysis of HRP, DAB produces Brown precipitation to realize signal amplification and color development.

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Package Contents:

Item code Description Volume
G1215-1 DAB diluent 12.5mL
G1215-2 50×DAB stock solution 250µL
G1215-3 HRP-conjugated Goat Anti-Rabbit IgG(H+L) 100µL
  User Manual 1pc

Technical Specification

Storage and Handling Conditions

Transport by wet ice; Secondary Antibody storage at -20°C; DAB diluent and 50×DAB stock solution storage at 4°C; Valid for 12 months.

 

Assay Protocol / Procedures

Preparation

  1. Self-prepared PBS (recommended G4202, G0002), Nucleus counterstaining reagent (recommended hematoxylin stain solution G1004, hematoxylin differentiation solution G1039 Hematoxylin returning blue solution g1040), gradient alcohol, xylene, sealing agent, etc.
  2. Preparation of secondary antibody working solution: HRP-conjugated Goat Anti-Rabbit IgG (H+L) was diluted with PBST (pH 7.2-7.4) in the ratio of 1:200 to obtain the secondary antibody working solution. Ready to use within 48 hours.
  3. Prepare DAB color developing working solution: Add 20μL 50× DAB stock solution to every 1 ml DAB diluent , mix well and set aside. Prepared on demand, ready to use.

Operation steps

  1. According to the routine IHC steps, after dewaxing, antigen repair, blocking and primary antibody incubation, the tissue sections were washed with PBS three times for 5 minutes each time.
  2. Secondary antibody incubation: add 50-100μL secondary antibody working solution on each slice,  completely covered the tissue and incubated at room temperature for 30-60 min. Wash three times with PBS for 5min each time.
  3. DAB chromogenic reaction: add 50-100μL DAB working solution on each section, incubated at room temperature for several minutes. The color developing time is controlled under the microscope. The positive is brownish yellow.Rinse the sections with tap water to stop the reaction.
  4. (OPTIONAL)Nucleus counterstaining: the sections are counterstained with hematoxylin stain solution for about 3 minutes; washed with tap water; differentiated with hematoxylin differentiation solution for several seconds; washed with tap water; treated with hematoxylin returning blue solution; washed with running water.
  5. Dehydration and mounting: place the section in 75% alcohol for 5 minutes--85% alcohol for 5 minutes--absolute ethanol 15 minutes--anhydrous ethanol 25 minutes--n-butanol 5 minutes--xylene 15 minutes, dehydrated and transparent, remove the sections from xylene and let them dry slightly, then mount the sections with neutral gum.

Note:

  1. If the background is too deep during the IHC chromogenic reaction, consider to extend the washing time, use appropriate blocking solution for mounting, inactive the endogenous catalase, shorten the chromogenic reaction time, reduce the concentration of secondary antibodies, etc. If there is no chromogenic reaction or the chromogenic reaction is too light, the concentration of primary antibodies and secondary antibodies can be properly increased, the chromogenic reaction time can be extended. Second, secondary antibodies were tested for normal color development.
  2. DAB is harmful to humans, when operating be careful, and be protected from direct contact or inhalation

 

For Research Use Only!

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