Reduction of background autofluorescence in brain sections following immersion in sodium borohydride

Coupling of Neuronal Nitric Oxide Synthase to NMDA Receptors via Postsynaptic Density-95 Depends on Estrogen and Contributes to the Central Control of Adult Female Reproduction

“…Coronal sections were cut at 80 m using a vibratome and processed for immunofluorescence. Sections were rinsed in PBS and reacted with 0.2% NaBH 4 for 45 min at RT to reduce tissue autofluorescence (Clancy and Cauller, 1998), rinsed again, and preincubated in blocking buffer (PBS containing 5% normal goat serum and 0.05% Triton X-100) overnight at 4°C. Primary antibodies (mouse anti-citrulline and rabbit anti-nNOS) diluted in blocking buffer were subsequently added to sections and incubated overnight at 4°C.…”

Treatment Strategies for the NMDA Component of Organophosphorus Convulsions

“…Treatment of the tissue sections with sodium borohydrate significantly reduced background autofluorescence induced by the formalin fixative and allowed clear visualization of ET fluorescence (Clancy and Cauller, 1998). As previously reported under normal physiological conditions ET fluorescence appears as small particles in the cytosol, suggesting mitochondrial generation of superoxide (Kondo et al, 1997;Murakami et al, 1998;Chan et al, 1998).…”

“…It is hypothesized that fixative aldehydes react with tissue amines to form fluorescent Schiffs bases (Clancy and Cauller, 1998). Sodium borohydrate acts to reduce the amine-aldehyde compounds into non-fluorescent salts, thereby reversing background autofluorescence (Clancy and Cauller, 1998).…”

“…Following postfixation the brains were rinsed thoroughly with PBS and sectioned into 50 nm sections by a vibratome. Brain sections were treated with 0.2% (w/v) sodium borohydride (Sigma) in PBS for 20 min under a ventilation hood and transferred into PBS for rinsing (Clancy and Cauller, 1998). Sections were mounted on glass slides with a coverslip using Gel/Mount mounting media (Fisher Scientific).…”

Triple-Labeling Whole-Mount In Situ Hybridization Method for Analysis of Overlapping Gene Expression in Brain Tissue with High Level of Autofluorescence

“…The color should appear orange under UV excitation, green or yellow under blue excitation, or red under green excitation. Several methods have been described for reducing autofluorescence: histochemical techniques (CuSO 4 in ammonium acetate buffer or Sudan Black B (SB) in 70% ethanol) [24]; NaBH 4 [25], Pontamine Sky Blue [26]. a multiple filter block (MFB) strategy, in which neurons are examined with three different filter blocks and a neuron is considered labeled if it fluoresces with only one of them; photobleaching by irradiation with ultraviolet (UV) light before treatment with fluorescent probes [27]; and mathematical models that subtract the background autofluorescence from digital images on a pixel-by-pixel basis [28].…”