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Paraformaldehyde (PFA) fixation and staining kit


Item Cat No.: BCASPF

Application: Immunohistochemistry

Reactivity: Tissue

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

1 Kit (100 slides)

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 with detergent 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 3.7% PFA fixation can preserve antigenicity better than 10% formalin fixation. PFA at 3.7% can also be used to perfuse animals to fix the medullar regions of many types of tissues.

The kit contains:

  • 50ml of Fixation buffer (3.7% PFA, NaCl, and phosphate buffered saline)
  • 100ml of university antigen retrieval buffer (proprietary)
  • 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 4oC

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

  • 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.
  • Dehydrate tissues with increasing concentrations of ethyl alcohol (70% to 100%).
  • Dehydrate tissues with Xylene, followed by embedding with paraffin.
  • Cut 5 μm thin paraffin sections.
  • Deparaffinize sections with Xylene.
  • Rehydrate sections with decreasing concentrations of ethyl alcohol (100% to 70%).
  • Wash slides 2 times with dH2O.
  • Dip slides into universal antigen retrieval buffer.
  • Microwave sections at boiling temperature for 10min.
  • Wash slides 2 times with dH2O.
  • Drain Wash solution against a towel and put slides into the wet chamber.
  • Dilute primary antibody at 1:50 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.
  • If using fluorophore labeled secondary antibodies, then 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.
  • If using HRP labeled secondary antibodies, then add HRP substrates such as DAB and follow the instructions of DAB colorimetric development.


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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

"The rabbit hybridoma supernatants of anti-APOBEC3 project are tested positive by ELISA, and we are very happy about it! We previously tried a company, Abclone. Their Project "A" has immune response that is <10,000 titer in antiserum, which would explain why there is no positive mAb after fusion. Their project "B" didn't have any immune response in rabbit."

Harshita B Gupta, PhD.

School of Medicine | UT Health San Antonio

"We have tested anti mouse T cell antiserum samples from both rabbits you sent to us.

They worked very well! Thank you!"

Victoria Gorbacheva, PhD.

School of Medicine | Cleveland Clinic

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