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Collagen fixation and staining kit

$99.00

Item Cat No.: BCASCO1

Application: 100 slides

Reactivity: Tissue, Cell culture

The collagen fixation and staining kit allows tissue fixation and immunolabeling of collagens with antibodies for IHC purposes.

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Extracellular matrix including collagen fibers is particularly difficult to fix and label with antibodies, largely because the strong molecular interactions within the matrix block the antibody access. BiCell Scientific has found that a combination of dehydrant and hydrogen bond breaker can effectively disrupt the molecular interactions in the extracellular matrix and expose the individual protein constituent for antibody recognition.

Dehydrants such as methanol and acetone remove and replace free water in the cell and tissue, and cause a change in the molecular interaction landscape of the extracellular matrix by destabilizing the hydrophobic interaction. Hydrophobic areas, frequently found on the inside of protein molecules, are exposed due to the repulsion of water, allowing antibody access to otherwise inaccessible protein domains.

Hydrogen bond breakers such as urea and acid can disrupt the hydrogen bonds made both between and within protein molecules that are vital for the high-order oligomeric structure of the extracellular matrix, such as collagen fiber. As a result, protein oligomers are dissociated into monomers, which greatly facilitates the antibody access and binding.

The kit contains:

  • 50ml of Fixation buffer (75% methanol and 25% acetone)
  • 15ml of Urea/Glycine buffer (Urea, glycine, and pH 3.5)
  • 50ml of Block buffer (bovine serum, Tween-20, NaCl and phosphate buffered saline)
  • 100ml of Wash buffer (Tween-20, NaCl, and phosphate buffered saline)
  • 1ml of Mounting buffer (Mowiol and Tris buffered saline)

Store at -20oC


For Research Use Only. Not for use in clinical diagnostics.

  • Thaw kit content (except Fixation buffer) to 4oC.
  • Snap freeze freshly dissected tissues with dry ice.
  • Cutting 5-10um cryostat sections.
  • Dip-fix sections into a jar filled with Fixation buffer at -20oC for 30 min.
  • Let slides air dry for 30min.
  • Dip slides into a jar filled with Urea/Glycine buffer. Fix at room temperature for 30min.
  • Wash slides three times with 1x PBS buffer.
  • Dilute primary antibody at 1:100 in Block solution and add to each slide (300-500 µl is sufficient to cover the section on each slide).
  • Incubate the slides in the wet chamber at room temperature for 2hr.
  • Take slides out of the wet chamber, drain off antibody, and wash three times with the Wash solution.
  • Dilute secondary antibodies at 1: 200 in Block solution and add to each slide and put slides back into the wet chamber.
  • Incubate the slides in the wet chamber at room temperature for 1hr.
  • Take slides out of the wet chamber, drain off antibody, and wash three times with the Wash solution.
  • Drain Wash solution against a towel. Add 20 ul of Mowiol solution to the section on each slide. Place a cover glass onto the section and apply a small pressure to remove any trapped air bubble.
  • Let Mowiol solution dry at room temperature for 1hr. Store glass slides at 4oC for less than 1 month.

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"I am really impressed with your approach. We tried multiple times previously to create monoclonal and polyclonal antibodies to claudin-2 and MLCK1. We have had limited success generating polyclonals and no success generating monoclonals. You have generated outstanding monoclonals to both. I look forward to continuing to work with you."

Jerrold R. Turner, M.D., Ph.D.

Brigham and Women’s Hospital | Harvard Medical School

"The polyclonal antibody you generated for KIAA0408 is stunning! KIAA0408 is a novel cilium molecule that has never been studied. So, clearly there will be a lot of demand for it as we have discovered a very interesting finding and the story will be published in a high impact journal. I am strongly inclined to generate monoclonal antibody for this protein too and we should think about patenting it."

Univ.-Prof. Jay Gopalakrishnan PhD

Heinrich-Heine-Universität | Universitätsklinikum Düsseldorf

"Your ARL13B antibody works beautifully!!! We’re so happy to have a cilia-specific antibody made in rat! I can send you high resolution images to be posted on your website."

Julie Craft Van De Weghe, PhD

School of Medicine | University of Washington

"The assay is a homophilic interaction mediated cell adhesion on purified protein (in this case, immobilized purified Pcdhga9 to Pcdhga9 expressed on cell surface). Compared to control, cell adhesion is reduced in the presence of Pcdhga9 monoclonal antibody supernatants!"

Divyesh Joshi, PhD

School of Medicine | Yale University

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