BN-PAGE gels were destained with 40% (v/v) methanol, 10% (v/v) glacial acetic acid for 30 min, refreshing the destaining solution every 10 min. Gels were soaked for 30 min in 1% SDS in Tris-buffered saline, pH 7.4, at room temperature. Proteins were blotted onto PVDF membrane by wet transfer at 100 V for 1 h. Coomassie-stained BSA marker bands were marked onto the membrane using a felt-tipped pen. Membranes were blocked in 20% (w/v) Marvel-skimmed milk powder in 0.1% PBS-Tween for 1 h. Membranes were incubated overnight at 4 °C on a roller in rabbit anti-AQP4 antibody (Abcam, ab128906) diluted 1:5,000 or rabbit anti-GFP (Abcam, ab6556) diluted 1:10,000, both in 5 ml of 0.1% PBS-Tween. Membranes were washed in 0.1% PBS-Tween and incubated with donkey anti-rabbit HRP (Santa Cruz, sc-2313) diluted 1:10,000 in 20 ml of 0.1% PBS-Tween at room temperature for 1 h. HRP was detected on x-ray film using ECL reagent (Amersham Biosciences).
Ab128906
Manufactured by Abcam
Ab128906 is a recombinant protein that functions as an antibody. It is produced using a baculovirus expression system. The product is supplied in PBS.
Automatically generated - may contain errors
4 protocols using ab128906
1
Western Blot Analysis of AQP4 and GFP
2
Cell Surface Protein Biotinylation and AQP4 Detection
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This was performed as described by Kitchen et al. (2015 ). Briefly, cell surface proteins were biotinylated using a cell‐impermeable amine‐reactive biotinylation reagent (EZ‐Link Sulfo‐NHS‐SS‐Biotin; Thermo Cat. No. 21331). 96‐well Pierce™ NeutrAvidin™ coated plates (Thermo Scientific; Cat. No. 15129) were used to pull down the biotinylated proteins within the total cell lysate. After washing off unbound proteins, the plate was then incubated with anti‐AQP4 antibody (Abcam, ab128906) diluted 1 : 500 in 0.05% PBS‐tween (PBS‐T), which bound to the cell surface biotinylated AQP4 proteins attached to the avidin coated plate. Anti‐AQP4 antibody validation data are provided in Fig. S1 .
3
Histopathological Analysis of Radiosensitive Tissues
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In this assay, the samples of radiosensitive, radioresistant, and ANT tissues were first fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Whereafter, 5-μm-thick sections were cut, followed by staining with hematoxylin and eosin for histopathology using a microscope (Olympus). For detecting the level of AQP4, the sections were washed with PBS and incubated with a primary antibody AQP4 (ab128906, 1:200 dilution; Abcam) at 4°C overnight. Finally, images were acquired for analysis.
4
Immunofluorescence Staining for mGluR5 and AQP4
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Immunofluorescence staining for mGluR5 and AQP4 was also performed on brain sections. Sections (4 μm) were blocked in 5 % bovine serum albumin and then subjected to overnight incubation using the anti-AQP4 (Cat.# ab128906, 1:100, Abcam) rabbit polyclonal antibody as well as the anti-mGluR5 (Cat.# ab76316, 1:200, Abcam) mouse polyclonal antibody at 4 °C and then 1 h of secondary antibody incubation (TRITC/Alexa Fluor 488-labelled anti-rabbit/mouse IgG) at ambient temperature. Sections were washed with PBS before being mounted with DAPI fluorescent mounting media. Using a fluorescence microscope (Carl Zeiss), the sections were photographed.
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