VitroView™ Immunofluorescence Double Staining Kit, FL594 Anti-Mouse (Red) & FL 488 Anti-Rabbit (Green) (For 50-100 slides)


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A double immunofluorescence (IF) procedure is needed in order to be able to examine the co-distribution of two different antigens in the same sample.

The advantages of this kit: 1) High sensitivity; 2) Low background; 3) Reduction of steps and time; 4) Simplified double labeling.


Unit Size 1 kit
Target Species Mouse and Rabbit
Color of Fluorescence FL594 Conjugated Goat anti Mouse IgG (Red)

FL488 Conjugated Goat anti Rabbit IgG (Green)

Excitation/Emission for FL488 dye 496/519 nm
Excitation/Emission for FL594 dye 590/617 nm
Host Species Goat
Application Double IF staining for simultaneous detection of primary antibodies raised in mouse and rabbit


RTU Normal Goat Serum ————————————————————-10ml

FL488 Conjugated Goat anti Mouse IgG (1mg/ml)————————–50µl

FL594 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

Storage Condition

Store at 2-8°C.


 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):

a. Fix in 10% formalin in PBS for 20 minutes (keep wet).

b. Immerse in ice-cold methanol for 10 minutes and allow to air dry.

c. 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:

a. 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.

b. 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.

c. 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.

d. 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 a mixture of two primary antibodies (mouse and rabbit IgG) 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 a mixture (70-150 µl) of two secondary antibodies raised in different species (with two fluorochromes including anti-mouse FL594 and anti-rabbit FL488 secondary antibodies) at appropriate dilutions (1/100-1/500) in 0.1% BSA in PBS for 1 hour at room temperature 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 and 594 nm.
  • Store slides in the dark at 2-8°C.

More Images

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

VB-6206 User Manual

VB-6206 MSDS


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.