Supplementary Figure 2 : Immunohistochemical identification of neuronal types.

From: Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing

Supplementary Figure 2

(a) Triple immunohistochemistry for LDHB, TRKB and TRKC and ISLET1. Note TRKB+-TRKC+/LDHB+ neurons (inset). All neurons belonging to the large Neurofilament, heavy polypeptide neuron class express Lactate dehydrogenase B (Ldhb). (b-e) Validation of the NF1 and NF2 TRKB+ subgroups. (b) Triple immunohistochemistry for CACNA1H, TRKB and ISLET1. Note TRKB+/CACNA1H+ neurons (inset). The Calcium channel, voltage-dependent, T type, alpha 1H subunit (CACNA1H) labels most of the neurotrophic tyrosine kinase, receptor, type 2, TRKB-expressing neurons. (c) Triple immunohistochemistry for NECAB2, TRKB and ISLET1. Note NECAB2+/TRKBhigh neurons (inset). The NF1 subgroup of neurons expresses the N-terminal EF-hand calcium binding protein 2 (NECAB2) and TRKB at high levels (TRKBhigh). (d) Triple immunohistochemistry for CALB1, TRKB and ISLET1. Note CALB1+/TRKBlow (inset). The NF2 subgroup expresses Calbindin (CALB1) and TRKB at low levels (TRKBlow, NF2). (e) Triple immunohistochemistry for NECAB2, CALB1 and ISLET1 shows NECAB2 and CALB1 being mutually exclusive (inset). (f-j) Validation of the NF3, NF4 and NF5 TRKC+ subgroups. The large, Neurotrophic tyrosine kinase, receptor, type 3 (TRKC) population is formed by 3 subgroups. (f) Triple immunohistochemistry for FAM19A1, TRKC and ISLET1. Note FAM19A1+/TRKC+ neurons (inset) and TRKC+/FAM19A1- neurons (arrowheads). NF3 is defined as a subpopulation of TRKC neurons expressing the Chemokine-like protein TAFA-1 (FAM19A1). (g) Triple immunohistochemistry for FAM19A1, Parvalbumin (PV) and ISLET1. Pvalb and Fam19a1 expression is mutually exclusive (inset). NF3 neurons are negative for Parvalbumin. (h) Triple immunohistochemistry for CNTNAP2, PV and ISLET1. Note PV+/CNTNAP2+ neurons (inset). (i) Triple immunohistochemistry for SPP1, CNTNAP2 and ISLET1. Note SPP1+/CNTNAP2+ neurons (inset) representing NF4 and NF5. NF4 and NF5 PV-positive neurons are a subpopulation of TRKC-positive neurons. (j) Triple immunohistochemistry for TRKC, CNTNAP2 and ISLET1. Note NF4 and NF5 CNTNAP2+/TRKC+ neurons (inset), subgroups of the largest TRKC population. We could not distinguish NF4 from NF5. However, Inhibin, beta B (INHBB) stood out as a good candidate to mark neurons belonging to the NF5 subgroup of TRKC+/PV+ neurons. (k) Triple immunohistochemistry for TH (sole marker defining TH population), combined TRKA/PLXNC1/NEFH and ISLET1. Note TH+/TRKA-/PLXNC1-/NEFH- neurons (inset). TH labels neither neurons belonging to the NF group (defined by Nefh expression), nor the NP group (defined by Plxnc1 expression) or the PEP group (defined by TrkA). (l-v) A combinatorial immunohistochemistry strategy was used to successfully distinguish the NP (l-p, v) and the PEP (q-u) classes of sensory neurons. Data analysis identified Plexin C1 (PLXNC1) as a common marker for all three subgroups of the NP group and, additionally, being expressed in some PEP1 neurons. (l) Triple immunohistochemistry for P2X3, PLXNC1 and SST. Note SST+/PLXNC1+/P2X3+ (inset). PLXNC1 labels all Somatostatin (SST) positive neurons belonging to NP3 and P2X3+ neurons belonging to NP1. (m) Triple immunohistochemistry for combined FAM19A1/TAC1, PLXNC1 and SST. Note (FAM19A1+ or TAC1+)/PLXNC1+/SST- (arrowheads) and (FAM19A1- and TAC1-) /PLXNC1+/SST+ neurons (inset). PLXNC1 expression is detected in NP3 and some neurons belonging to PEP groups. (n) Triple immunohistochemistry for FAM19A1, PLXNC1 and CGRP. Fam19a1 and Plxnc1 expression is mutually exclusive (inset); PEP2 neurons do not express PLXNC1 (o) Triple immunohistochemistry for TAC1, PLXNC1 and CGRP. Note TAC1+/PLXNC1+/ CGRP+ neurons (inset); expression patterns of PLXNC1 and of Tachykinin, precursor 1 (TAC1), a unique marker of PEP1 neurons, show some overlap. (p) Triple immunohistochemistry for PLXNC1, combined FAM19A1 and TAC1 and CGRP, shows CGRP+/FAM19A1-TAC1-/PLXNC1+ neurons belonging to the NP2 subgroup. (q) Triple immunohistochemistry for CGRP, TRKA and ISLET1. TRKA and CGRP show 1:1 co-localization (inset). The Neurotrophic tyrosine kinase, receptor, type 1 (TRKA) and Calcitonin receptor-like (CGRP) are expressed by all neurons belonging to NP2, PEP1 and PEP2 subgroups. (r) Triple immunohistochemistry for TAC1, TRKA and ISLET1. TAC1 defines PEP1 neurons. Note TRKA+/TAC1+ (inset) and TRKA+/TAC1- neurons (arrowheads) belonging to the other two TRKA-expressing groups, NP2 and PEP2, respectively. (s) Triple immunohistochemistry for FAM19A1, TRKA and NEFH. Note NEFH+/FAM19A1+/ TRKA+ and NEFH+/FAM19A1+/ TRKA- neurons, belonging to PEP2 and NF3, respectively (inset) and TRKA+/FAM19A1- neurons (arrowheads) belonging to the other two TRKA-expressing groups (NP2 and PEP1). (t) Triple immunohistochemistry for combined FAM19A1/TAC1, TRKA and ISLET1. Note TRKA+/FAM19A1-/TAC1- neurons (inset and arrowheads), belonging to NP2 subgroup. (u) Triple immunohistochemistry for SST, TRKA and ISLET1. Sst and TrkA expression is mutually exclusive (inset). Sst defines the NP3 subgroup. (v) Double immunohistochemistry for PLXNC1 and combined NFH/TRKA/SST/TH. Note PLXNC1+/NFH-/TRKA-/SST-/TH- neurons belonging to the NP1 subclass. Scale bar = 50 μm.