Aqp 004

Heat-induced antigen retrieval was done on the paraffin embedded sections by boiling the sections in Tris-EGTA buffer, pH 9.0 (CD45), TRS buffer (Target Retrieval Solution, DAKO) (F4/80), or TEG buffer, pH 9.0 (AQP4), first 15 min at 900W, then 9 min at 440W. The sections were allowed to cool in the buffer before blocked for endogenous peroxidase and biotin activity. Sections were then incubated with anti-CD45 (1:100; clone 30-F11 (Ly 5); BD Pharmingen), anti-F4/80 (1:100, AbD Serotec), or anti-AQP4 (1:1250; AQP-004, Alomone labs) antibodies and detected using biotinylated rabbit anti-rat IgG (DAKO) (CD45 and F4/80) or biotinylated donkey anti-rabbit IgG diluted 1:200 followed by ready-to-use anti-rabbit horse-radish perioxidase (HRP)-labelled polymer (EnVision + System, DAKO) with diaminobenzidin (DAB⁺) as chromogen (DAKO). Nuclei were counterstained using Mayer’s haemalum w/4.5% chloralhydrate. As negative control, the primary antibody was omitted to check for any unspecific reaction from the detection system. As positive control for antibody-specificity, the staining was tested using a mouse multi block containing several different tissues including lymphatic organs. The activation state of CD45⁺, and F4/80⁺ microglia and leukocytes and AQP4 expression were investigated in 5 sections (representing 750 μm spinal cord) from each animal centered on the lesion epicenter.

Whole spinal cord protein samples from α₂^+/G301R and α₂^+/+ littermate mice exposed to SCI and allowed 3 days survival in addition to naïve α₂^+/G301R and α₂^+/+ littermate mice were prepared as described [50 (link)]. Equal amounts of protein were separated by SDS-PAGE on 10–14% (α_1, α₂, α₃ and AQP4) gels and electro-blotted onto nitrocellulose membranes (Pharmacia-Amersham). Membranes were blocked in PBS with 5% skimmed milk and 0.5% Tween-20 and incubated with the following primary antibodies: anti-α₁ diluted 1:2000 (clone a6f-c, Developmenal Studies Hybridoma Bank), anti-α₂ diluted 1:1000 (Merck Millipore), anti-α₃ diluted 1:1000 (Merck Millipore), anti-AQP4 diluted 1:1000 (AQP-004, Alomone labs) anti-GAPDH diluted 1:1000 (Abcam), or anti-β-Actin diluted 1:2000 (Sigma-Aldrich) overnight at 4 °C.
Next, membranes were incubated with HRP-conjugated secondary antibodies (swine anti-rabbit HRP diluted 1:2000 (Dako) or rabbit anti-mouse HRP diluted 1:2000 (Dako)) for 1 h at room temperature. Visualization of blots was done in a LAS 3000 imager (Fujifilm) with Amersham ECL Western Blotting Detection Kit (GE Healthcare). Post densitometric analysis and image processing of blots were performed in Image J.

Sheep anti rat megalin (Moestrup et al. 1993 (link)), rabbit anti rat AQP4 (AQP-004) (Alomone Labs, Jerusalem, Israel), rabbit anti rat AQP1 (AB 2219) (Merck Millipore, Billerica, MA, USA), rabbit anti rat NaK Atpase (α₂ isoform) (AB 9094) (Merck Millipore) and goat anti human NKCC1 (SC-21545) (Santa Cruz Biotechnology, Dallas, TX, USA).