Fig 1: Human glossopharyngeal/vagal nerve at the level of medulla oblongata. The tissue contained both cross sectional cuts of the nerves (a-c & g-i) and longitudinally cut sections of the nerve (d-f). Six micron thin sections were stained for ACE2 (a & d) using a rabbit primary antibody (diluted 1:350) and amplifying it with Tyramide-Alexa-594 (Red). Next a rabbit primary antibody to TMPRSS2 (b & e) was applied (diluted 1:100) followed by a Tyramide-Alexa-488 amplification (green). At the end, a rabbit primary antibody to fibronectin (c & f) was applied (diluted 1:5000) followed with an anti-mouse Alexa-647 (colored yellow) secondary antibody. The right upper inset in a-f shows the overlays of the antibodies in the same areas. A separate section was stained with a rabbit primary antibody to Fibronectin (h) followed by an anti-mouse Alexa-647 (colored yellow) secondary antibody and finally an anti-Neuropilin (g) pre-conjugated to Alexa-594 was used (diluted 1:100). (i) shows the overlay of NRP1 and Fibronectin. Cell nuclei are stained with DAPI (blue). *All primary antibodies were applied overnight. *All secondary antibodies were diluted 1:500.
Fig 2: Human glossopharyngeal/vagal nerve at the level of medulla oblongata. The tissue contained both longitudinally cut sections of the nerve (a-d & i-l) and cross-sectional cuts of the nerve (e-h & m-p). Six micron thin sections were stained for ACE2 using a rabbit primary antibody (diluted 1:100) and an anti-rabbit Alexa-594 (red) conjugated secondary antibody (a&e) next they were stained for neurofilament using a chicken primary antibody (diluted 1:10000) (b, f, j, n) followed by either anti-chicken Alexa-488 (green) (b&f) or anti-chicken Alexa-555 (Red) (j&n). Another set of sections were stained first with anti-neuropilin1 (NRP-1) pre-conjugated to Alexa-488 antibody (diluted 1:100) (i&m), followed by neurofilament staining (j&n) visualized by anti-rabbit Alexa-594 (red) conjugated secondary antibody. Finally, all sections were stained using an anti-Myelin Basic Protein (MBP) (c,g,k,o) pre-conjugated to Alexa-647 (Diluted 1:100) (colored white). (d,h,l,p) shows the overlay of the three antibodies. All cell nuclei are stained with DAPI (blue). *ACE2 & NRP-1 antibodies were applied overnight at 4°C, MBP was applied for two hours at RT. *All secondary antibodies were diluted 1:500. Scale: 20 µm from a-l and 40 µm from m-p.
Fig 3: Immuno-stained cultured oligodendrocytes and CNS fibroblasts. Cells grown in chamber slides were stained for ACE2, NRP-1 and TMPRSS2 using human immortalized oligodendroglioma cells and human primary brain vascular fibroblast. Chamber slides of each cell line were stained with either ACE2 (a, d) using a rabbit primary antibody and an Alexa-594 (red) conjugated secondary antibody, TMPRSS2 (c, f) using a rabbit primary antibody and an anti-rabbit Alexa-594 (red) conjugated secondary antibody, or an anti-Neuropilin (b, e) pre-conjugated to Alexa-488 antibody. Cell nuclei are stained with DAPI (blue). *All primary antibodies were applied overnight with a 1:100 dilution. *All secondary antibodies were diluted 1:500.
Fig 4: RT-PCR of human frozen nerve. Fresh human nerves (N) were collected and treated with Trizol for RNA preparation. RNA was reverse transcribed for the nerves and human lung (L) RNA purchased as a positive control. RT-PCR was performed to test the presence of NRP1(137bp), TMPRSS2 (106bp and 158bp) and ACE2 (124bp, and 199bp). For normalization, β-actin (ACTB) was used as a housekeeping gene (184bp), and a 100bp ladder was used for size verification. Two primer sets were used for both the ACE2 and the TMPRSS2 {10]. Negative control (-) (no template) does not show any bands.
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