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Complete IHC kit for Cryosection

$650.00$750.00

Item Cat No.: BCASKC

Application: IHC on cryosection

Reactivity: Tissue

BiCell Scientific’s complete IHC kit for cryosection provides a full set of reagents including two internal control antibodies for immunolabeling of cryosections using fluorescent or chromogenic detection method.

1 Kit (100 slides)

Dehydrants such as methanol and acetone remove and replace free water in the 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.

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. BiCell Scientific’s complete IHC kit for cryosection provides a full set of reagents and protocols including two internal control antibodies, rabbit anti E-cadherin polyclonal antibody and rat anti ZO-1 polyclonal antibody, for immunolabeling of cryosections using fluorescent or chromogenic detection method.

The kit components (fluorescent option):

  • 50 ul of anti E-cadherin antibody (rabbit polyclonal)
  • 50 ul of anti ZO-1 antibody (rat polyclonal)
  • 50 ul of anti rabbit secondary antibody (Alexa Fluor 488)
  • 50 ul of anti rat secondary antibody (Alexa Fluor 594)
  • 100ml of Fixation buffer (75% methanol and 25% acetone)

Store at –20oC

  • 100 ml of Block buffer (bovine serum, Tween-20, NaCl and Tris buffered saline)
  • 500 ml of Wash buffer (Tween-20, NaCl, and Tris buffered saline)
  • 1 ml of Antifade-Fluorescence Mounting Medium

Store at 4oC


The kit components (enzyme-substrate option):

  • 50 ul of anti E-cadherin antibody (rabbit polyclonal)
  • 50 ul of anti ZO-1 antibody (rat polyclonal)
  • 50 ul of anti rabbit secondary antibody (Horseradish peroxidase)
  • 50 ul of anti rat secondary antibody (Alkaline phosphatase)
  • 100ml of Fixation buffer (75% methanol and 25% acetone)

Store at –20oC

  • 100 ml of Block buffer (bovine serum, Tween-20, NaCl and Tris buffered saline)
  • 500 ml of Wash buffer (Tween-20, NaCl, and Tris buffered saline)
  • 50 ml of DAB substrate for Horseradish peroxidase
  • 50 ml of BCIP/NBT substrate for Alkaline phosphatase
  • 10 ml of Antifade-Chromogen Mounting Medium

Store at 4oC


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

Fluorescent detection:

  • Thaw kit content (except Fixation buffer) to 4oC.
  • Snap freeze freshly dissected tissues with dry ice.
  • Cutting 5-10 μm 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.
  • Add 20 ul of Antifade-fluorescence mounting medium to the section on each slide. Place a cover glass onto the section and apply a small pressure to remove any trapped air bubble. Air-dry at room temperature for 1hr. Store glass slides at 4oC for less than 1 month.

 


Enzyme-substrate detection:

  • Thaw kit content (except Fixation buffer) to 4oC.
  • Snap freeze freshly dissected tissues with dry ice.
  • Cutting 5-10 μm 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.
  • Apply 100 ul of substrate solution to tissue section until desired color is achieved (1-10 mins).
  • Rinse off substrate with PBS.
  • Dehydrate sections with increasing concentrations of ethyl alcohol (70% to 100%).
  • Dehydrate sections with Xylene. Air-dry in fume hood for 30min at room temperature.
  • Add 100 ul of Antifade-chromogen mounting medium to the section on each slide. Place a cover glass onto the section and apply a small pressure to remove any trapped air bubble. Air-dry at room temperature for 1hr. Store glass slides at ambient temperature.

Additional information

Detection

Fluorescence, Enzyme-Substrate

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Be the first to review “Complete IHC kit for Cryosection”

"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

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