Alexa fluor 488 conjugated donkey anti mouse igg antibody

One hour after restraint, skin samples were taken from the wrist, which most frequently showed Evans blue dye leakage in the hypertensive rats. The skin samples were paraffin-embedded, sectioned (5 μm), and incubated with primary antibodies (1:500, mouse anti-CGRP; Chemicon, USA; Cat# MAB317, RRID:AB_2275141), followed by incubation with secondary antibody (1:500, Alexa Fluor 488-conjugated donkey anti-mouse IgG antibody, Thermo Scientific; Cat# A11055, RRID:AB_142672). The sections were mounted on gelatine-coated slides, air-dried, and coverslipped with a mounting medium containing DAPI (Vector, Cat# H-1500). The sections were examined using a laser-scanning confocal microscope (LSM700, Carl Zeiss, Germany).

Three days after BDL surgery, rats (n = 6) were given EBD injection and neurogenic spots (n = 6) and non-neurogenic spots 5 mm apart from the neurogenic spots (Non-Neuro-Sp, n = 6) were taken. The skin samples were paraffin-embedded, sectioned at 5 μm thickness and incubated with either anti-SP mouse monoclonal antibodies (1:500; GeneTex International Corporation, Irvine, CA, USA) or anti-CGRP mouse antibodies (1:500; Chemicon International Inc., CA, USA), followed by incubation with secondary antibody (1:500, Alexa Fluor 488-conjugated donkey antimouse IgG antibody, Thermo Scientific, Waltham, MA, USA). The sections were mounted on gelatin-coated slides and cover-slipped. Skin images were taken from three sections from each skin with an epifluorescence microscope (Olympus BX51, Tokyo, Japan) and quantified by using ImageJ software (National Institute of Mental Health, Bethesda, MD, USA). The mean intensity of green fluorescence was measured.

Staining procedures were performed as previously described.² (link) In short, skeletal muscles were labeled by submersion in the following solutions, all diluted in 1x PBS unless otherwise specified: 0.1 M glycine for 10 min to reduce tissue autofluorescence; 1x PBS wash for 10 min; 2-h permeabilization in 5% Triton X-100; 30-min blocking in 4% bovine serum albumin and 2% Triton X-100. Primary antibody (in block solution) incubation was then completed over 60 h at 4°C with mouse anti-SV2 (synaptic vesicles; 1:50 dilution; DSHB, Iowa City, Iowa) and mouse anti-2H3 IgG (neurofilaments; 1:50 dilution; DSHB).
Tissues were washed in 1x PBS for 4 × 20 min and then incubated in the secondary antibody, 2 μg/mL AlexaFluor-488-conjugated donkey anti-mouse IgG antibody (Cat: A21202, Thermo Scientific, Waltham, Massachusetts) in 1x PBS diluted 1:500, and left overnight at room temperature. Samples were kept in the dark from this stage to prevent photobleaching. A further 4 x 20-min washes in 1x PBS were followed by 15–30 min (muscle dependent) in 2 μg/mL tetramethyl-rhodamine isothiocyanate-conjugated α-bungarotoxin (acetylcholine receptors; Cat: BTIU00012, VWR International, Radnor, Pennsylvania). Muscle fibers were mounted in Mowoil on glass slides for imaging.