Ready to use high-efficiency immunohistochemical secondary antibody kit,Absin_specification/price/image

Product Detail
Company Profile

Product Specification

Usage

1. Following the conventional Western blot operation, after the second antibody is incubated, during the last washing, according to the size of the membrane, mix 0.5ml of solution A and 0.5ml of solution B every 10 cm2 of the membrane, and prepare a luminescent detection working solution. 2. Use flat headed tweezers to remove the membrane, with the lower edge of the membrane gently touching the absorbent paper to remove excess liquid from the membrane. The protein side of the film should be facing upwards and placed on a clean cling film (some commercially available cling films may quench fluorescence when wrapping imprinted films, so high-quality cling films should be chosen). Transfer the prepared luminescent detection solution onto the protein film using a pipette, cover it evenly, and incubate at room temperature for 2 minutes. 3. Use flat headed tweezers to grip the protein film, gently touching the lower edge of the film with absorbent paper to remove excess liquid from the film. The protein side of the film is facing upwards and wrapped in a clean cling film. Gently push out the bubbles between them and fix them in the X-ray cassette. 4. Take an X-ray film in the darkroom and place it on the wrapped film. Close the cassette and expose it for 30 seconds to 1 minute. Immediately develop and fix, shorten or extend the exposure time of the next X-ray film based on its exposure intensity (for weak signals, the exposure time can be extended to several hours).

Protocol

1. According to the routine Western blot operation, after the incubation of the secondary antibody and the last wash, according to the size of the membrane, 0.5ml solution A and 0.5ml solution B were mixed per 10 cm2 membrane to prepare the luminescence detection working solution.
2 Remove the membrane with flat-tipped tweezer and gently touch the lower edge of the membrane with absorbent paper to remove excess liquid from the membrane. The protein side of the membrane is facing up and placed on clean plastic wrap (some commercial plastic wrap may quench fluorescence when wrapping the imprinted film, and high-quality plastic wrap should be selected). The prepared luminescent detection solution was transferred to the protein membrane with a pipette so that it was uniformly covered and incubated for 2 min at room temperature.
3. Hold the protein membrane with a flat-headed tweezer, and the lower edge of the membrane gently touches the absorbent paper to remove excess liquid on the membrane. Membrane protein on the face, wrapped in a clean plastic wrap. Gently expel the air bubbles in between and fix them in the X-ray cassette.
4. Take an X-ray film in the dark room and place it on the wrapped film, close the cassette, and expose it for 30 seconds to 1 minute. "The image is developed immediately, and the exposure time of the next radiograph is shortened or extended depending on its exposure intensity (for weak signals, the exposure time can be extended to several hours).

Description

packaging: 5ml (can be used for 50 pieces)

applicable: primary antibody is from mouse or rabbit

chromogenic substrate: dab (benzidine)

reagent composition:

component number	component name	specification	quantity
A	Hydrogen peroxide	5ml	1
B	Super sealing fluid	5ml	1
C	Primary antibody amplification agent	5ml	1
D	Enzyme labeled secondary antibody polymer	5ml	1
E	Dab a liquid	5ml	1
F	DAB B fluid	160ul	1

reagents not provided but required: primary antibody (from mouse or rabbit), PBS (phosphate buffer)

this kit is the latest generation of non biotin detection system for immunohistochemical secondary antibody based on polymer technology, with stronger signal and simpler operation. Compared with the traditional immunohistochemical secondary antibody kit, it has three characteristics: first, due to the uniqueness of the polymer, the polymer molecule formed by the direct combination of enzyme molecules and secondary antibody IgG molecules is highly sensitive, effectively reducing the amount of primary antibody used, and the primary antibody reagent can be diluted 2-4 times; Second, because the use of biotin is avoided, the non-specific background color development caused by biotin can be avoided, and a clearer color development and cleaner background can be obtained; Third, rapid and shorter reagent incubation time

storage and validity period

2-8° C save. Each component can be stored for at least 18 months

operation steps:

note: all steps are operated at room temperature. Adding reagent can be titrated directly in a drop bottle or with a pipette. One drop =30-40ul

1 Dewaxed and hydrated tissue sections

2 Wash 2-3 times with PBS for 5 minutes each

3 According to the special requirements of the applied primary antibody, the tissue sections were pretreated

4 Wash 2-3 times with PBS for 5 minutes each

5 Add 100ul A solution was incubated for ten minutes to block endogenous peroxidase to reduce nonspecific background staining

6 Wash 2-3 times with PBS for 5 minutes each

7 Add 100ul B solution was incubated for five minutes to reduce nonspecific staining. Note: this step should not exceed ten minutes; If the primary antibody is diluted in a buffer containing 5% to 10% normal goat serum, this step can be omitted

8 Wash 2-3 times with PBS for 5 minutes each

9 Add primary antibody and incubate at room temperature or 37 ℃ for 20 min

10 Wash 2-3 times with PBS for 5 minutes each

11 Add 100ul C solution incubation for ten minutes

12 Wash 2-3 times with PBS for 5 minutes each

13 Add 100ul Incubate with D solution for ten minutesNote: this liquid is sensitive to light, and pay attention to avoiding light

14 Wash 2-3 times with PBS for 5 minutes each

15 Prepare fresh substrate solution: add 30ul of F liquid and 1ml of E solution mix well to prepare a fresh substrate solution (the substrate solution can be stored for 2 weeks)

16 Add 100ul of fresh substrate solution and incubate for five minutes

17 Deionized water flushing

18 Counterstain, seal

Component

Liquid A; liquid B

AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.