Exalpha Biologicals, Inc.

Accelerating the Pace of Discovery

Product Highlight

FIX&PERM Cell Fixation and Permeabilization Kit

Flow cytometric analyses with monoclonal antibodies were so far mainly restricted to cell surface molecules. Intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins etc. were largely excluded from such studies. Also excluded from flow cytometric studies were cytoplasmic localizations of well-established membrane molecules like CD3 and CD22, which, in their cytoplasmic form, are the most reliable lineage markers in undifferentiated leukemia. With the FIX&PERM® Kit flow cytometric analysis of intracellular antigens has become as easy as surface antigen studies. The only prerequisite is the availability of suitable antibody conjugates. Most of the available monoclonal antibody conjugates can be used with the FIX&PERM® Kit, some determinants are sensitive, however, to the fixation step involved. This and the optimal fixation time have to be tested for each reagent.

News

Two more of our excellent products have been published by PubMed:

Potential actionable targets in appendiceal cancer detected by immunohistochemistry, fluorescent in situ hybridization, and mutational analysis
Borazanci, E., et al., J. Gastrointest. Oncol., 8, 164-172 (2017)
Using Exalpha SPARC Antibody (Cat. No. X1867P)

Molecular mechanism underlying the pharmacological interactions of the protein kinase C-β inhibitor enzastaurin and erlotinib in non-small cell lung cancer cells
Steen, N.V., et al., Am. J. Cancer Res., 7, 816-830 (2017)
Using Exalpha's FITC labeled anti PY20 Antibody (Cat. No. X1017)

Helpful Hints for Using Exalpha Immunofluorescence Kits

Issue

Tip


Autofluorescence

  1. One method of checking for autofluorescence is to look at the slide with multiple filters. If the same signal is seen with multiple filters, it could be autofluorescence.
  2. To help decrease autofluorescence, add a Sudan Black step after the SA-FL incubation.
    1. Make up the Sudan Black at 0.2% in 70% Ethanol (mix for 2 hours in the dark).
    2. After the SA-FL incubation and wash step, add 100 µL of 0.2% Sudan Black to the slide.
    3. Incubate for 15 minutes at room temperature in the dark.
    4. Wash 8 X 1 minute using the wash buffer recommended in the assay specific protocol.
    5. Mount and view the slide under the microscope.

Supressed Signal

If Sudan Black was used:
  1. While the use of Sudan Black can help decrease autofluorescence, it can also decrease signal. Sudan Black should be used with caution.
  2. The addition of 0.02% tween to the wash step following the Sudan Black incubation may help to increase the signal.
  3. Sudan Black should be made up fresh each time.
  4. Sudan Black can be tested at at a lower concentration (e.g., 0.025%).
Additional tips:
  1. Completed slides should be stored in the dark when they are not in use.
  2. Slides should be analyzed shortly after the mounting media is dry.

The signal is
difficult to find

  1. Use the bright field setting on the microscope to focus in on the correct section of the tissue prior to using the excitation wavelength listed in the kit specific protocol.
  2. Make sure that the work area surrounding the microscope is dark.
  3. Allow your eyes to adjust to the dark prior to viewing the slides.