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Non-aggregating High-resolution Secondary Antibody Labeling of Primary Cilia

Non-aggregating High-resolution Secondary Antibody Labeling of Primary Cilia

Sequential sections of mouse retina were stained with anti-NPHP5 antibody (Cat No: 90005), followed by labeling of two different secondary antibodies conjugated with Alexa Fluor 488. Top panel: BiCell Scientific's secondary antibody; bottom panel: Thermo Fisher's secondary antibody.

Non-aggregating High-resolution Secondary Antibody Labeling of Cell Junction

Non-aggregating High-resolution Secondary Antibody Labeling of Cell Junction

Sequential sections of mouse colon were stained with anti-ZO-1 antibody (Cat No: 00236), followed by labeling of two different secondary antibodies conjugated with Alexa Fluor 594. Top panel: BiCell Scientific's secondary antibody; bottom panel: Thermo Fisher's secondary antibody.

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Non-aggregating High-resolution Secondary Antibody Fluorophore Conjugate

$375.00

Antibody: Goat Secondary Polyclonal Antibody

Concentration: 0.50 mg/ml

Application: IF/IHC on Cryosection and FFPE

Reactivity: Rabbit, Rat, Mouse

200 µl size

Non-aggregating High-resolution Secondary Antibody Fluorophore Conjugate allows fluorescent labeling of primary polyclonal or monoclonal antibodies.

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Fluorophore conjugated secondary antibodies allow location studies of target proteins at subcellular to cellular levels with nanometer resolution.

The most common approach for fluorophore conjugation to secondary antibody is by succinimidyl ester derivative of fluorophores such as FITC or Alexa Fluor dyes. However, fluorophore conjugation alters the solubility of antibody molecules, rendering aggregation of secondary antibodies at subcellular levels, which significantly reduces the spatial resolution of fluorescent signals.

BiCell Scientific Inc has developed a proprietary conjugation approach that is able to maintain the solubility of fluorophore conjugated antibodies, thus minimizing the aggregation of secondary antibody molecules and improve the spatial resolution of fluorescent signals.

Host/Isotype: Goat/IgG

Class: Polyclonal

Immunogen: Target species IgG (H+L)

Conjugation: Fluorophore

Purification: Affinity chromatography

Storage buffer: PBS, pH 7.2, 0.1% sodium azide

Storage condition: –20°C

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

  • Deparaffinize FFPE 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.
  • Dip slides into FFPE autofluorescence quenching buffer for 20min.
  • Wash slides with 70% ethyl alcohol
  • Wash slides with 1xPBS
  • 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.
  • 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.

Additional information

Target species

Rabbit, Rat, Mouse

Fluorescent labels

Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647

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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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