Glial fibrillary acidic protein (gfap)

We used the mouse PSMA/FOLH1/NAALADase I/GCPII antibody raised against Lys44-Ala750 which contains the extracellular domain of GCPII (MAB4234; R&D Systems; 1:50). The antibody recognizes a very specific band for GCPII by western blotting with no other non-specific bands. The recognition of PSMA/FOLH1/GCPII was confirmed using antigen-down ELISA (R&D Systems). In direct ELISA’s, the antibody shows less than 10% cross-reactivity with rhNAALADase-like 2 and no cross-reactivity with rhNAALADase-like 1, rhNAALADase-like 3, rmNAALADase I, or rmNAALAase-like 2. The multiple label immunofluorescence used the following additional antibodies: GFAP (1:500, BioLegend, Cat# 829401), NeuN (1:300, EMD Millipore, Cat# ABN78), and Iba1/AIF-1 (1:300, Cell Signaling Tech, Cat# 17198 T).

Cultured cells or brain slices (30 μm) were fixed with 4% paraformaldehyde followed by three washes in PBS and permeabilized with 0.1% Triton X-100 for 10 min (except for O4, O1 immunostainning) then blocked in 10% goat serum (Invitrogen Corp. USA) for 1 h following incubation with the primary antibody overnight at 4 °C. Primary antibodies were diluted in blocking solutions as follows: mouse anti-MBP (Biolegend, USA; 1:500), mouse anti-APC, CC1 clone (Millipore, USA; 1:500), mouse anti-O4 (R&D Systems Inc, USA, 1:800), mouse anti-O1 (R&D Systems Inc, USA, 1:800), rabbit anti-PDGFRα (Cell Signalling Technology, USA; 1:500), rabbit anti-glial fibrillary acidic protein (GFAP, Biolegend, USA; 1:500) mouse anti-BrdU (Sigma St Louis, USA; 1:500). The tissue or cells were then washed with PBS and incubate in Alexa Flour-conjugated secondary antibodies (Invitrogen Corp. USA; 1:500) for 1 h. Images were obtained using Olympus photomicroscope and analyzed using Image-Pro plus 6.0 software.

4d after brain slice preparation the tissue was fixed and pre-embedded in 2% agar (Carl Roth # 5210.3)- 2.5% gelatin (Merck Millipore # 1040700500; in PBS) without sponge pads and subsequently processed for paraffin embedding. 4-6 μm thick sections were stained according to standard immunohistochemistry protocols. The antibodies used were specific for: glial fibrillary acidic protein (GFAP) (Biolegend # 644701; diluted 1:500), laminin (Progen Biotech # 10765; diluted 1:200), myelin basic protein (MBP) (Abcam # 7349; diluted 1:200) and ionized calcium-binding adapter molecule 1 (Iba1) (Wako # 019–19,741; diluted 1:500).

For SC dissociation and isolation, paired MPGs (n = 4 rats) were excised, digested in collagenase/dispase/hyaluronidase, and dissociated by gentle trituration. Dissociated SCs were suspended in a SC medium containing Dulbecco’s Modified Eagle Medium with d-valine (HiMedia, West Chester, PA) as previously described.¹¹ (link) Once confluent, SCs were plated onto individual glass coverslips coated with laminin and poly-l-lysine, covered with SC medium, and again incubated to confluence for 24-48 hours. Dissociated neurons from control and diabetic rats were plated on top of the confluent SC and cocultured for 48 hours in a 50:50 mixture of neurobasal and SC media which was changed every 24 hours. After 48 hours, cocultured neurons and SCs were fixed and stained with primary antibodies specific for TUJ1 (1:100, rabbit; Sigma-Aldrich), GFAP (1:200 anti-mouse; BioLegend, San Diego, CA), or S100 (1:200; anti-chicken, MyBioSource, San Diego, CA) captured as previously described, and the longest neurite from each neuron was measured and counterstained with DAPI.

PFA fixed cells or 6-µm formalin fixed paraffin-embedded sections were stained according to standard immunohistochemistry protocols. The following antibodies were used: mCherry (abcam, ab167453), Iba1 (Wako, 01919741), Cd44 (Abcam 119863), CD31 (Abcam, ab28364), GFAP (BioLegend, 644701). Secondary antibodies were from Thermo Fischer Scientific. For brain tissue analysis, images were acquired with Zeiss Axio-Scan.Z1 using ZEN software (Zeiss, Oberkochen, Germany). Cultured cells were imaged with a MEA53100 Eclipse Ti-E inverted microscope (Nikon, Japan). Analysis were performed using ImageJ.