Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue

Niteace C. Whittington1, Susan Wray1

1 Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.28
DOI:  10.1002/cpns.35
Online Posting Date:  October, 2017
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Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc.

Keywords: autofluorescence; red blood cell; fluorescent microscopy; embryonic tissue; immunocytochemistry

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Litrature Cited
  • Figures
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Basic Protocol 1:

  • Timed‐pregnant mouse
  • 70% ethanol
  • Phosphate‐buffered saline (PBS), pH 7.4 ± 0.2 (see appendix 2A)
  • 4% (w/v) formaldehyde in PBS (e.g., Avantor, cat. no. 5016‐02)
  • 30% (w/v) sucrose in PBS
  • Tissue‐Plus O.C.T. Compound (e.g., Fisher Scientific)
  • Citrate buffer (see recipe)
  • NHS/Triton: 10% (v/v) normal horse serum (NHS)/0.3% (v/v) Triton X‐100
  • Primary Antibodies:
    • Rabbit anti‐Peripherin (1:3000; e.g., Millipore, cat. no. AB1530, RRID:AB_90725)
    • Rabbit anti‐Pax6 (1:400; e.g., Millipore, cat. no. AB5409, RRID:AB_2315065)
    • Goat anti‐Sox21 (1:100; e.g., R and D Systems, cat. no. AF3538, RRID:AB_2195947)
    • Guinea pig anti‐DCX (1:3500; Millipore, cat. no. AB5910, RRID:AB_2230227)
    • Rabbit anti‐GnRH‐1 (SW­1, 1:15,000; see Wray, Gahwiler, & Gainer, )
  • Secondary antibodies (1:1000):
    • Alexa Fluor 488 donkey anti‐rabbit IgG (e.g., Thermo Fisher Scientific, cat. no. A21206, RRID:AB_2535792)
    • Alexa Fluor 555 donkey anti‐rabbit IgG (e.g., Thermo Fisher Scientific, cat. no. A31572, RRID:AB_162543)
    • Alexa Fluor 488 donkey anti‐goat IgG (e.g., Thermo Fisher Scientific, cat. no. A11055, RRID:AB_2534102)
    • Alexa Fluor 488 donkey anti‐guinea pig IgG (e.g., Jackson ImmunoResearch Labs, cat. no. 706‐545‐148, RRID:AB_2340472)
  • 20× TrueBlack Lipofuscin Autofluorescence Quencher in DMF (e.g., Biotium, cat. no. 23007)
  • Vectashield Antifade Mounting Medium with DAPI (e.g., Vector Laboratories, cat. no. H‐1200)
  • CO 2 chamber and source
  • Forceps
  • Small surgical scissors
  • Tissue culture dishes
  • 12‐well plate
  • Foil boxes (12 × 12 × 10 mm)
  • Cryostat
  • Charged super‐frost microslides
  • Staining box
  • Glass coverslips
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.
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Literature Cited

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