Cy3 conjugated affinipure goat anti mouse igg h l

The tissues of rainbow trout were dissected and fixed in 4% neutral buffered formalin overnight at 4°C, embedded in paraffin, and 4 μm thick sections stained with hematoxylin and eosin (H&E) or alcian blue (AB) as described previously (13 (link)). Images were acquired in a microscope (Olympus) using the Axiovision software. For the detection of IgT⁺ and IgM⁺ B-cells, sections were double stained with polyclonal rabbit anti-trout IgT (pAb; 0.5 μg ml^–1) and monoclonal mouse anti-trout IgM (IgG1 isotype; 1 μg ml^–1) overnight at 4°C. After washing three times, sections were stained with Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) and Cy3-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson ImmunoResearch Laboratories Inc.) at 2.5 μg ml^–1 each for 40 min at room temperature to detect IgT⁺ and IgM⁺ B-cells, respectively. For detection of pIgR⁺ cells in trout BM, we used the same methodology described previously by using polyclonal rabbit anti-pIgR antibody (pAb; 0.8 μg ml^–1) (18 (link)). Before mounting, all sections were stained with DAPI (4’, 6-diamidino-2-phenylindole; 1 μg ml^–1; Invitrogen). All images were acquired and analyzed using an Olympus BX53 fluorescence microscope (Olympus) and the iVision-Mac scientific imaging processing software (Olympus).

The impact of the noise exposure on the bank of hippocampal stem cells in DG region was observed at 3 and 7 days post noise exposure (3DPN and 7DPN) by counting the BrdU+ cells. BrdU was given at the dose of 50 mg/kg 4 h before the animals were sacrificed. The hippocampal slices were stained first with an antibody against BrdU {1:500, Anti-BrdU antibody [BU1/75(ICR1)], Rat monoclonal, Abcam, ab6326} at 4°C overnight, and followed by the treatment with the second antibody [1:1000, Donkey Anti Rat IgG H&L (Alexa Flour 488^®), Abcam, ab150153] at 37°C for 1 h. The slice was then treated with a solution containing the first antibodies against Nestin and GFAP (1:100, Anti-Nestin, clone rat-401, Mouse monoclonal, Millipore, MAB353; 1:250, Anti-GFAP antibody, Rabbit polyclonal, Abcam, ab7260)at 4°C overnight, followed by a mixture of the second antibodies [1:1000, Cy 3-conjugated AffiniPure Goat Anti-Mouse IgG H&L, Jackson, 115-165-166; 1:500, Donkey anti-Rabbit IgG (H + L) secondary Antibody, Alexa Fluor^® 647 conjugate, Life Invitrogen, A-31573]. The triple positive cells (BrdU+/Nestin+/GFAP+) were counted as the stem cells.