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Methanol fixation and staining kit


Item Cat No.: BCASME

Application: IF on cryosection

Reactivity: Tissue, Cell culture

The methanol fixation and staining kit allows tissue fixation with methanol and immunolabeling with antibodies for IHC purposes.

Dehydrants such as methanol and acetone remove and replace free water in the cell and tissue, and cause a change in the tertiary structure of proteins by destabilizing the hydrophobic interaction and the hydrogen bonds. 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.

Methanol fixation is particularly useful for polymeric or oligomeric protein structures, such as extracellular matrix, cell junction complex, and focal adhesion complex. Because methanol can partially dissolve lipids, methanol fixation offers better antibody access to the cytoplasm of the cell. The disadvantage is that the morphology of plasma membrane is not well preserved.

Because acetone is a stronger solvent for lipids and a stronger denaturant for proteins than methanol, BiCell Scientific has found that a 1:3 mixture of acetone with methanol allows better fixation and antibody labeling than methanol alone.

The kit contains:

  • 50ml of Fixation buffer (75% methanol and 25% acetone)
  • 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.
  • 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

I have tested the rat polyclonal IgGs to ABCD1 by immunoflourescence on cells overexpressing ABCD1. The antibodies successfully detected the protein (either untagged or tagged with GFP) at a 1:500 dilution and there was little background. The antibodies did not detect ABCD2. So this is very good news, and you may now go ahead and clone out a monoclonal!

Annette Ehrhardt, PhD

Dept. of Pediatrics | Emory University

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