Tissue Immunostaining and Quantification

Tissue immunostaining was performed, as previously described.^{84 (link)} Tissue sections and cell cultures were fixed by 4% PFA in PBS for 30 min, permeabilized in 0.3% Triton X-100 for 1.5 h at room temperature, and blocked in 4% normal serum (Jackson ImmunoResearch Laboratories, West Grove, PA) for 20 min. Primary antibodies specific for CA8, V5, vinculin, TrkA, pTrkA (Abcam, Cambridge, MA), pITPR1 and ITPR1 (Cell Signaling Technology, Danvers, MA). Antibodies were allowed to incubate with sections and cell cultures overnight at 4°C after dilution in PBS containing 0.1% Triton X-100. Sections and cultures were washed three times for 10 min each in PBS, and incubated with Alexa Fluor 488 and/or Alexa Fluor 594-conjugated second antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at RT. Sections and cultures were mixed with the 2^nd antibody to assess the total number of cells, and images were acquired as described elsewhere.^{24 (link)}