Ab62024

Antibodies against reticulon 4B (AB-163; Kinasource Ltd, Dundee, UK), reticulon 4A (ab62024; Abcam, Cambridge, UK), HA (MMS-101R-50; Covance, Princeton, NJ), FLAG (F7425; Sigma-Aldrich), calreticulin (2679S; Cell Signalling Technologies, MA) and β-actin (ab8227-50; Abcam) were used as primary antibodies. When indicated, rabbit anti-sheep bridging antibody (313-001-003; Jackson ImmunoResearch Labs Inc., West Grove, PA) was used. Secondary antibodies were Rhodamine Red-X (016-290-084; Jackson ImmunoResearch), Alexa 647 (A31571; Life technologies), Alexa 488 (A-11008; Life Technologies) and 1.4 nm nanogold-conjugated anti-rabbit antibody (Nanoprobes, Stony Brook, NY).

The rats (n = 4 per group) without undergoing MRI experiments were deeply anesthetized. The perilesional cortex was separated, and protein levels were determined by Western blotting, as previously described (Zhan et al., 2020 (link)). Proteins were transferred onto polyvinylidene difluoride membranes, followed by blocking them with 5% nonfat milk for 2 h and subsequently incubating membranes at 4°C overnight with primary antibodies: anti-GAP-43 (1:40000; Epitomics, #2259-1), SYN (1:320000; Epitomics, #1870-1), Netrin-1 (1:2,000; Abcam, ab126729), DCC (1:1,000; Abcam, ab125280), Slit-2 (1:10,000; Abcam, ab134166), Robo-1 (1:1,000; Abcam, ab7279), NogoA (1:20,000; Abcam, ab62024), NgR (1:40,000; Abcam, ab62024), RhoA (1:20,000; Cell signaling, 2117s), ROCK-2 (1:50,000; Abcam, ab125025), and GAPDH (1:1,60,000; GeneTex, GTX627408). After washing, membranes were incubated with secondary anti-rabbit (1:20,000; Applygen Technologies Inc., C1309) or anti-mouse (1:20,000; NeoBioscience, cat. ANM 02-1, Lot. 0912) IgG (H + L)-HRP for 1 h at room temperature. Immunoreactive protein bands were detected by using the SuperECL Plus kit (Applygen, China, cat. No. P1050) and chemiluminescent imager (VILBER, United States). The intensities of target proteins were quantified by ImageJ software.

Immunofluorescence double labeling was performed as previously described.¹³ The primary antibodies (Ab) were goat anti‐Olig2 (1:200 dilution, AF2418, R&D Systems), rabbit anti‐Olig2 (1:200 dilution, ab109186, Abcam), rabbit anti‐Nogo‐A (1:100 dilution, ab62024, Abcam), and goat anti‐LCN2 (1:100, AF1757, R&D Systems). The secondary Abs were Alexa Fluor 488‐conjugated donkey anti‐rabbit mAb (1:500 dilution, Invitrogen), Alexa Fluor 488‐conjugated donkey anti‐goat mAb (1:500 dilution, Invitrogen), Alexa Fluor 594‐conjugated donkey anti‐rabbit mAb (1:500 dilution, Invitrogen), and Alexa Fluor 594‐conjugated donkey anti‐goat mAb (1:500 dilution, Invitrogen). Negative controls omitted the primary antibodies.
Olig2 is a helix–loop–helix transcription factor found in all OPCs, immature oligodendrocytes, and mature oligodendrocytes. Thus, Olig2⁺ cell numbers reflect all oligodendrocytes. In contrast, Nogo‐A identifies only the mature phenotype. Cells which were Nogo‐A⁻/Olig2⁺ cells were subclassified as oligodendrocyte precursor cells/immature oligodendrocytes.¹⁶, ²⁶, ²⁷