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VitroView™ Anti-Rabbit Immunofluorescenc Staining Kit (Green, 50-100 slides)

$110.00

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Description

Immunofluorescence (IF) is a vital immunochemical technique enabling the detection and precise localization of numerous antigens across diverse tissue types and various cell preparations. The VitroView™ immunofluorescence single staining Kit is a reliable and user-friendly solution designed for use with primary antibodies specifically raised in rabbit.

Key Advantages:

  1. Enhanced Sensitivity: Achieve high sensitivity in antigen detection, enhancing the quality of your results.
  2. Minimal Background Interference: Reduce background noise to a minimum, ensuring clear and reliable data.
  3. Streamlined Workflow: Save time and effort with a simplified procedure that minimizes the number of steps required.
  4. Easy Multiple Labeling: Facilitate multiple labeling experiments, making your research more efficient.

Specifications

Unit Size 1 kit
Target Species Rabbit
Conjugates and Color of Fluorescence FL488 Conjugated Goat anti Rabbit IgG (Green)
Excitation/Emission for FL488 dye 496/519 nm
Host Species Goat
Application Immunofluorescence staining for detecting a primary antibody made in rabbit

Contents

  • RTU Normal Goat Serum ——————————————-10ml
  • FL488 Conjugated Goat Anti-Rabbit IgG (1mg/ml)————–50µl
  • Aqueous Anti-fade Mounting Medium with Dapi—- 1.5 ml×2

Note: RTU=ready-to-use

Reagents and Material Required but Not Provided

  • Xylene and ethanol
  • Distilled or deionized water
  • 10 mM phosphate-buffered saline (PBS), pH 7.4
  • Triton X-100
  • Mini PAP Pen
  • Primary antibody
  • Antibody Dilution Buffer (SKU#: VB-6002)
  • BSA
  • Antigen retrieval reagents

Storage Condition

Store at 2-8°C.

Protocol

 1. Preparation of Slides

A. Cell Lines

Grow cultured cells on sterile glass cover slips or slides overnight at 37°C. Briefly wash with PBS. Choose one of the following fixation methods (To achieve optimal results, it may be necessary to optimize the fixation methods):

  • Fix in 10% formalin in PBS for 20 minutes (keep wet).
  • Immerse in ice-cold methanol for 10 minutes and allow to air dry.
  • immerse in ice-cold acetone for 10 minutes and allow to air dry.

Wash slides in PBS.

 B. Frozen Sections

  • Snap-freeze fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, then embed them in OCT compound in cryomolds. Store the frozen tissue block at -80°C until ready for sectioning.
  • Before sectioning, equilibrate the frozen tissue block to the temperature of the cryotome cryostat (e.g., -20°C).
  • Section the frozen tissue block into the desired thickness (typically 5-10 µm) using the cryotome.
  • Place the tissue sections onto glass slides suitable for immunohistochemistry (e.g., Superfrost).
  • Store sections in a sealed slide box at -80°C for later use.
  • Before staining, allow slides to warm to room temperature for 30 minutes and then fix in ice-cold acetone or ice-cold methanol for 10 minutes. Air dry for 30 minutes.
  • Wash in PBS.

C. Paraffin Sections

  • Deparaffinize sections in xylene, 3×5 minutes.
  • Hydrate with 100% ethanol, 2×2 minutes.
  • Hydrate with 95% ethanol, 2×2 minutes.
  • Rinse in distilled water.
  • Follow the required pretreatment procedure.

2. Antigen Retrieval

Most formalin-fixed tissues require antigen retrieval before proceeding with immunohistochemical staining. Common methods include heat-mediated and enzymatic antigen retrievals.

Choose the appropriate method:

  • For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0, and maintain at a sub-boiling temperature for 10 minutes. Cool slides on the benchtop for 30 minutes.
  • For EDTA: Bring slides to a boil in 1 mM EDTA, pH 8.0, and follow with 15 minutes at a sub-boiling temperature. No cooling is necessary.
  • For TE: Bring slides to a boil in 10 mM TE/1 mM EDTA, pH 9.0, and then maintain at a sub-boiling temperature for 18 minutes. Cool at room temperature for 30 minutes.
  • For Pepsin: Digest for 10 minutes at 37°C.

Note: Do not use this pretreatment with frozen sections or cultured cells that are not paraffin-embedded.

3. Staining Procedure

  • Rinse sections in PBS-Triton X-100 (0.025%) for two 2-minute cycles.
  • Serum Blocking: Incubate sections with 2-4 drops of ready-to-use (RTU) normal goat serum for 30 minutes to block non-specific immunoglobulin binding.
  • Primary Antibody: Incubate sections with primary rabbit antibody at the appropriate dilution in antibody dilution buffer (SKU# #: VB-6002) for 1-2 hours at room temperature or overnight at 2-8°C. Rinse in PBS.
  • Detection: Incubate sections with FL488-conjugated Goat Anti-Rabbit IgG secondary antibody at suitable dilutions (ranging from 1/100 to 1/500) in 0.1% BSA in PBS for 1 hour at room temperature while keeping the samples in the dark.
  • Rinse in PBS for three 2-minute cycles.
  • Counterstaining and mount cover slip with 20-30µl of aqueous anti-fade mounting medium with DAPI. Seal cover slip with nail polish to prevent drying and movement.
  • Visualize and capture the signal using a fluorescence microscope equipped with excitation wavelength filters of 488 nm.
  • Store slides in the dark at 2-8°C.

More Images

User Manual and Material Safety Data Sheet (MSDS)  (PDF)

VB-6103 User Manual

VB-6103 MSDS

Note:

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

Precautions:

Handle with care. Avoid contact with eyes, skin and clothing. Do not ingest. Wear gloves.