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PFA fixation and staining kit


Item Cat No.: BCASPF

Application: IF on cryosection

Reactivity: Tissue, Cell culture

The paraformaldehyde (PFA) fixation and staining kit allows tissue fixation with PFA and immunolabeling with antibodies for IHC purposes.

Paraformaldehyde crosslinks are formed between protein molecules, in particular with the basic amino acid – lysine. Only those lysine residues on the exterior of the protein molecule can react with paraformaldehyde. Paraformaldehyde crosslinks are made primarily on the hydrophilic areas of proteins. The reaction between aldehyde and protein is pH-dependent, progressing more rapidly at higher pH values.

Paraformaldehyde is the condensation product of methylene glycol. It is a white solid with a formaldehyde content between 78 and 98%. Paraformaldehyde can be depolymerized to formaldehyde solution by water in the presence of a base and heat. In water, formaldehyde is slowly hydrated to form a glycol at acidic pH.

Residual formaldehyde must be quenched by primary amine such as Tris and Glycine to reduce the autofluorescence in tissue samples. Formaldehyde does not act on lipids in the cell membrane. A permeabilizing step is needed to facilitate antibody access to the cytoplasm.

Paraformaldehyde concentrations ranging from 3.7-4% are often used in fixation buffer. BiCell Scientific has found that lower paraformaldehyde concentrations such as 1-2% can better preserve antigenicity while still retaining good tissue morphology. Low paraformaldehyde content can reduce the osmotic shock to cells as well.

The kit contains:

  • 50ml of Fixation buffer (2% PFA, NaCl, and phosphate buffered saline)
  • 50ml of Sucrose buffer (30% sucrose and phosphate buffered saline)
  • 50ml of Block buffer (bovine serum, Tween-20, NaCl and Tris buffered saline)
  • 100ml of Wash buffer (Tween-20, NaCl, and Tris 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 to 4oC.
  • Perfuse animal with Fixation buffer. Cut tissues into small blocks (~5 mm cubic shape) using a scalpel. Dip-fix tissue blocks in Fixation buffer at 4oC for 8-12hr.
  • Transfer tissue blocks to Sucrose buffer and dip-fix at 4oC for 8-12hr.
  • Cutting 5-10um cryostat sections.
  • Dip-fix sections into a jar filled with Fixation buffer at 4oC for 10 min.
  • Wash slide 2 times with Wash buffer.
  • Drain Wash solution against a towel and put slides into the wet chamber.
  • 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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