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Sirius Red/Fast Green Collagen Staining Kit

Product description

Item Name: Fast Sirius Red / Green Collagen Staining Kit

Biozol catalogue number: CHX-9046

Supplier Catalog Number: 9046

Alternate Catalog Number: CHX-9046

Manufacturer: Chondrex

Category: Kits / Assays

Product properties

Range: Collagen: 3 µg / section Other proteins: 50 µg / section

Storage: RT

Introduction

Sirius Red specifically binds to the [Gly-X-Y] n helical structure of fibrillar collagens such as type I to V collagen and is used to detect all types and species of collagen, while Fast Green binds non-collagen proteins. Exploiting the unique characteristics of these two dyes, Chondrex provides a simple semi-quantitative assay kit to determine the amounts of collagen and non-collagen proteins in cultures cell layers and tissue sections. Because this assay does not require collagen solubilization, it is widely used for the measurement of total collagen content in various tissues.

Since Sirius Red and Fast Green have absorptions at 540 nm and 605 nm respectively, the OD values ​​of the extracted dyes can be used to calculate the content of collagen and non-collagen proteins in each section. For general histological studies in which tissue sections are made at a thickness of 10-20 µm, the sensitivity of the assay for collagen and non-collagen proteins is greater than 3 µg / section and 50 µg / section, respectively. This kit contains enough reagents to stain between 30 and 50 samples.

Test Procedure

The following sample preparation methods are standard protocols. Therefore, these protocols may need to be optimized depending on the sample types.

  • Paraffin-embedded tissue sections

1. Prepare paraffin-embedded tissue sections (approximately 30-50 nm2, 10-20 μm thick).
2. Deparaffinize the tissue section with the following steps below:

  • Xylene, 10 minutes
  • Xylene 1: 1 with 100% ethanol, 10 minutes
  • 100% ethanol, 10 minutes
  • 50% ethanol / distilled water, 5 minutes
  • Distilled water, 5 minutes

3. Transfer individual slides to Petri dishes.
4. Add 0.2-0.3 ml of dye solution to completely submerge the tissue section and incubate at room temperature for 30 minutes.

5. Gently remove the dye solution.
6. Rinse the stained tissue section with 0.5 ml of distilled water repeatedly until the liquid is colourless.
7. In a Petri dish, add 1 ml of dye extraction buffer and mix gently by pipetting until the colour is eluted from the tissue section.
8. Collect the eluted dye solution and read the OD values ​​at 540 nm and 605 nm with a spectrophotometer.

  • Frozen tissue sections

1. Prepare frozen tissue sections (approximately 30-50 nm2, 10-20 μm thick) according to standard methods.
2. Wash with PBS.

Kahle’s Stationary Prescription (Contact Customer Service to purchase this tampon):

  • 60 ml of distilled water
  • 28 ml of 96% ethanol
  • 10 ml of 37% formaldehyde
  • 2 ml of concentrated acetic acid

3. Remove the fixative and wash with PBS, then transfer individual slides to Petri dishes.
4. Add 0.2-0.3 ml of dye solution to completely submerge the tissue section and incubate at room temperature for 30 minutes.

5. Gently remove the dye solution.
6. Rinse the stained tissue section with 0.5 ml of distilled water repeatedly until the liquid is colourless.
7. In a Petri dish, add 1 ml of dye extraction buffer and mix gently by pipetting until the colour is eluted from the tissue section.
8. Collect the eluted dye solution and read the OD values ​​at 540 nm and 605 nm with a spectrophotometer.

  • In vitro cultured cell layers

1. If you plan to mount cell layers and observe under a microscope before proceeding to the dye extraction step, place sterile round glass slides at the bottom of each well of a 24-well culture plate. Otherwise, you can skip this step.
2. Grow cells in the 24-well culture plates for a desired period of time.
3. Remove the culture medium and wash the wells with PBS.
4. Add 0.5 ml of Kahle fixative to completely submerge the tissue section and incubate for 10 minutes at room temperature.

Kahle’s Stationary Prescription (Contact Customer Service to purchase this tampon):

  • 60 ml of distilled water
  • 28 ml of 96% ethanol
  • 10 ml of 37% formaldehyde
  • 2 ml of concentrated acetic acid

5. Remove the fixative and wash with PBS.
6. Add 0.2-0.3 ml of staining solution to completely submerge the fixed cell layers and incubate at room temperature for 30 minutes. If the cells are on glass slides, remove the slides and place them in Petri dishes for staining.
7. Gently remove the dye solution

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