JCS031047 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. NMJ-capping cells are Thy-1 negative but colocalise after denervation with activated terminal Schwann cells. Panels showing combined (left) and separated (middle and left) colour channels of preparations immunostained with 2166 antibody and either Thy 1.1 (A) or S100 (B). (A) Co-immunostaining of mouse triangularis sterni muscle with 2166 antibody (green) and Thy-1.1 antibody (red) show no colocalisation. However, Thy-1 immunopositivity was observed in some of the other perijunctional fibroblasts and intramuscular capillaries. Acetylcholine receptors were counterstained with fluorescent bungarotoxin (pseudocoloured blue). (B) Co-immunostaining of rat fourth deep lumbrical muscle with 2166 antibody (green) and S100 antibody (red) 6 days after section of the tibial nerve. Sprouts between muscle fibres made by NMJ-capping cells and terminal Schwann cells are clearly coincident and associated, but the antibodies are evidently not colocalised in the same cells.

• Supplemental Figure S2 -

  Fig. S2. Antibody 2166 immunostaining of transverse muscle sections shows thin NMJ-capping cell processes lying outside the synaptic basal lamina, as indicated by co-staining for laminin (red) and Alexa Fluor 647-α-BTX (blue). Supplementary to Fig. 3B,C. Scale bar: 20 µm

• Supplemental Figure S3 -

  Fig. S3. High-resolution image of the NMJ shown in Fig. 4D. Note the thin basal lamina around most of the putative NMJ-capping cell and its processes above the NMJ, and the presence of rough ER in the cytoplasm. MF, muscle fibre; NT, nerve terminal; tSC, terminal Schwann Cell; K, kranocyte; BL, basal lamina. Scale bar: 2 µm.

• Supplemental Figure S4 -

  Fig. S4. Co-staining of TS muscles in unoperated muscles (A,B) and 1 day after axotomy (C,D) with 2166 antibody (green), DAPI (blue; nuclei) and TRITC-α-BTX (AChR). These images show evidence of a rapid increase in the number of 2166-positive cells per NMJ following denervation. Scale bar: 30 µm (A,B); 20 µm (C,D).

• Supplemental Figure S5 -

  Fig. S5. Co-staining of a 3-day denervated TS muscle with 2166 antibody (green), nestin (red) and Alexa Fluor 647-α-BTX (blue). Note the association of a cell processes from an NMJ capping cell and a nestin-positive tSC sprout. This figure shows separation of the RGB channels for the same image as Fig. 5E. Scale bar: 40 µm.

• Movie 1 -

  Movie 1. Z-series of triple-stained NMJ shown in flat projection in Fig. 2B rPH (red); 2166 (green); AChR (blue)

• Movie 2 -

  Movie 2. Animated rotating projection of z-series for triple-stained NMJ shown in Movie 1 and in flat projection in Fig. 2B. rPH (red); 2166 (green); AChR (blue)

• Movie 3 -

  Movie 3. Z-series of endplate AChR stained with TRITC-α-BTX, shown in projection and with volume rendering in Fig. 3D

• Movie 4 -

  Movie 4. Z-series of NMJ-capping cells stained with CTB, shown in projection and with volume rendering in Fig. 3D

• Movie 5 -

  Movie 5. Animated rotating projection of Z-series of NMJ-capping cells stained with CTB, shown in Movie 4 and in projection with volume rendering in Fig. 3D

• Movie 6 -

  Movie 6. Volume-rendered animation of a reconstructed NMJ-capping cell shown in Fig. 4A-C.

• Movie 7 -

  Movie 7. Volume-rendered animation of the other reconstructed NMJ-capping cell shown in Fig. 4A-C.