Axioscope 2 fluorescence microscope

Parasites were fixed in 1% paraformaldehyde for 10 min at room temperature (RT). Parasites were washed in 1X PBS and adhered to slides spread with Poly-L-lysine before being permeabilised with 0.1% Igepal in 1X PBS for 3 min. Cells were then blocked with 2% BSA in 1X PBS for 45 min at RT, stained with primary antibody (anti-PAD1^{35 (link)} 1:1000, EP1 procyclin [Cedar labs] 1:300) diluted in 0.2% BSA for 1 h at RT. Three washes with 1X PBS were performed before incubating with secondary Alexa Fluor 488 (ThermoFisher Scientific) in 0.2% BSA for 1 h at RT. Cells were washed a further three times before mounting with Fluoromount G with DAPI (Cambridge Bioscience, Southern Biotech). Imaging was performed with an Axioscope 2 fluorescence microscope (Zeiss) and a Zeiss Plan Apochromat 63x/1.40 oil objective. Image analysis was carried out with Fiji ImageJ v2.

Fluorescent dye ATTO Fluor 590-conjugated MT7 (MT7-ATTO590; Alomone Labs) was used to detect M₁R. The activity of this MT7-ATTO590 conjugate on M1R was confirmed by the company (Alomone Labs) in M₁R/C6 cells by measuring intracellular changes in Ca²⁺ levels and the specific binding was determined in rat DRG culture in the presence of excess (1 µM) unlabeled MT7 (data not shown). Adult wild-type and M₁R-KO (C57BL/6 background, line 1784; Taconic Biosciences Inc.) [46 (link)] mouse DRG tissues were incubated with 100 nM MT7-ATTO590 containing media at 37 °C CO₂ incubator overnight and then fixed in 2% PFA, cryoprotected in 20% sucrose, and embedded in Tissue-Tek O.C.T. compound to prepare 7 µm sections. All sections were incubated overnight at 4 °C with β-tubulin III antiserum (1:500; T8578, Sigma) and then stained for 1 h with Alexa Fluor 488-conjugated anti-mouse IgG (1:1000; Invitrogen, California, USA) at room temperature. To confirm M₁R expression in SH-SY5Y cells and cultured rat DRG neurons, cells/neurons were incubated with 100 nM MT7-ATTO590 at 37° C in a CO₂ incubator overnight for microscopy. All images were taken by using a Carl Zeiss LSM510 confocal or Axioscope-2 fluorescence microscope.