One for All—A Highly Efficient and Versatile Method for Fluorescent Immunostaining in Fish Embryos

Abstract

BackgroundFor the detection and sub-cellular (co)-localization of proteins in the context of the tissue or organism immunostaining in whole mount preparations or on sections is still the best approach. So far, each antibody required its own fixation and antigen retrieval protocol so that optimizing immunostaining turned out to be tedious and time consuming.Methodology/Principal FindingHere we present a novel method to efficiently retrieve the antigen in a widely applicable standard protocol, facilitating fluorescent immunostaining of both cryosections and whole mount preparations in zebrafish (Danio rerio) and medaka (Oryzias latipes).Conclusions/SignificanceOur method overcomes the loss of sections and damage of tissue and cell morphology, and allows parallel immunostaining in multiple colors, co-immunostaining with fluorescent proteins in transgenic fish lines and in combination with whole mount in situ hybridization.

TRPV4-Mediated Detection of Hyposmotic Stress by Skin Keratinocytes Activates Developmental Immunity

“…Tg(krt18:RFP) larvae were fixed in 4% paraformaldehyde, dehydrated in methanol/PBS, and stored in 100% methanol at 220˚C. For staining, larvae were rehydrated in methanol/PBS and 0.1% Tween 20 (PBT), washed in PBT, incubated with 150 mM Tris-HCl (pH 9), followed by heating at 70˚C for 15 min (27). After heating, larvae were washed in PBT and dH 2 O.…”

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

“…False‐colored and superimposed overviews of 24 hpf embryos were analyzed for spontaneous movement assays. Whole‐mount fluorescent immunostaining were carried out as described in (Inoue and Wittbrodt, 2011), embedded in JB‐4 (Polysciences) and 5 µm sections were cut. As primary antibodies we used: polyclonal rabbit anti‐GFP (1:200, Thermo Fisher Scientific, #A11122) and monoclonal mouse anti‐α‐actinin (1:10, Sigma Aldrich, #A7811).…”

Loss of zebrafish Smyd1a interferes with myofibrillar integrity without triggering the misfolded myosin response

“…Whole-mount RNA in situ hybridization was carried out essentially as described previously [6] using a full-length smyd1b antisense probe, as well as antisense probes for zebrafish smyd1a, vmhc, amhc, hsp90aa1 and unc45b. Whole mount fluorescent immunostainings were carried out as described in Inoue and Wittbrodt [9]. Fish were embedded in JB-4 (Polysciences) and 5 mm sections were cut.…”