Donkey anti mouse alexa 594

Fluorescent immunohistochemistry was performed on cryostat-sectioned brains. Sections were washed in PBS followed by primary antibody incubation overnight. All antibodies were diluted in supermix. Anti-MFSD1 (1:50) and anti-MFSD3 (1:50) were used to label the proteins. They were co-stained with the neuronal nuclear marker anti-NeuN (1:400 mouse, cat. no: MAB377, Millipore) (Mullen et al. 1992 ), the astrocyte marker anti-GFAP (1:400 mouse, cat. no: MAB360, Millipore) (Reeves et al. 1989 (link)) and the dendritic marker anti-MAP2 (1:400 mouse, cat. no: M4403, Sigma-Aldrich) (Izant and McIntosh 1980 (link)). After additional PBS washes, slides were incubated with secondary antibody for 2 h at RT. Secondary antibodies used are as follows: alexa 488 goat-anti-rabbit, alexa 488 donkey-anti-goat and alexa 594 donkey-anti-mouse (Invitrogen) diluted 1:400. PBS washes followed by 10 min DAPI staining (1:3000 in PBS, Sigma-Aldrich) and additional washes before mounted in Mowiol anti-fade media (25 g Mowiol 4–88 in 100-ml 1× PBS, pH 8.0, 50 ml glycerol, 3 ml of 1 % thimerosal and 100 μg/ml n-propyl gallate in (Sigma-Aldrich)). Images were taken using an Olympus microscope BX53 with an Olympus DP73 camera. The micrographs were acquired by cellSens Dimension software.

To explore whether neurons in the NTS projecting to the ACC were activated by painful CP, double immunostaining of FG and FOS was performed in the NTS of CP rats 1 week after FG injection into the ACC. TNBS was administered 1 week before FG injection to establish the CP model. The preparation of brain sections was as previously described. After confirmation of the injection site in the ACC and virus-labeled cell bodies in the NTS, immunostaining was performed using the following antibodies and procedures: after blocking, the ACC sections were incubated in mouse anti-FOS (1:500; ab11959, Abcam) and guinea pig anti-FG (1:200, NM-101, ProtosBiotech, New York, NY, USA) overnight at 4 °C. The sections were then incubated with Alexa594-donkey anti-mouse (1:500; A21203, Invitrogen) or Alexa488-donkey anti-guinea pig (1:500; 706-545-148, Jackson Immunoresearch, West Grove, PA, USA) for 4 h at 4 °C in darkness. The expression of FOS in FG-labeled neurons in the NTS was observed under the confocal microscope.

Fluorescent immunohistochemistry was performed on 7 μm paraffin embedded coronal sections. The sections were rehydrated before antigen retrieval through 10 min boiling in 0.01mM citric acid (Sigma-Aldrich), pH 6.0. The sections were washed in PBS, before addition of primary antibodies, diluted in 5% milk blocking solution (Blotting grade blocker, Bio-Rad). Anti-MFSD4A (1:50) and anti-MFSD9 (1:50) were co-stained with anti-NeuN (1:400, mouse, Millipore, MAB377) and anti-GFAP (1:400, mouse, Millipore MAB360). Subsequently, the sections washed in PBS before incubated with the secondary antibodies Alexa 488 goat-anti-rabbit, Alexa 488 donkey-anti-mouse, Alexa 594 donkey-anti-mouse and Alexa 594 donkey-anti-goat (Invitrogen), diluted 1:800. The sections were mounted in Mowiol anti-fade mounting medium before imaged in an Olympus fluorescence microscope BX53, with an Olympus DP73 camera. The micrographs were acquired by cellSens Dimension software and show representative staining.

Immunohistochemistry was performed on brains collected from ChR2+ and ChR2− mice (see Supplementary Methods 3.1). Primary antibodies used were mouse monoclonal anti-TPH2 (1:500; Sigma-Aldrich) and chicken anti-GFP (1:1000; Abcam). Secondary antibodies used were donkey anti-mouse Alexa 594 (1:1000; Invitrogen) and donkey anti-chicken Alexa 488 (1:1000; Jackson Immunoresearch). Sections were imaged using a confocal microscope.

Cells were seeded on Nunc Lab-TekII CC2 Chamber Slides (Thermo-Fisher Scientific). After 16 h treatment with 1 µM XAV939 or DMSO, cells were washed with PBS, fixed in PBS-buffered 4% paraformaldehyde for 10 min, permeabilized with PBS-0.2% Triton X-100 solution for 5 min and blocked with PBS containing 3% BSA for 30 min. Samples were incubated with primary antibodies at room temperature for 1 h, followed by PBS-Tween 20 0.05% washes and incubation with appropriate secondary antibodies for 1 h. Primary antibodies were diluted as follows: AXIN1 (AF3287, 1:100); AXIN2 (#2151, 1:200); Tankyrase-1/2 (sc-365897, 1:200). The following secondary reagents were used: Donkey anti-Goat-Alexa 647 (#A-21447), Donkey anti-Rabbit-Alexa 488 (#A-21206), Donkey anti-Mouse-Alexa 594 (#A-21203); all from Invitrogen, at 1:500 dilution. Slides were mounted with Vectashield mounting medium with DAPI (H-1200, Vector Laboratories). Images were generated using a Zeiss LSM510META confocal electroscope.

Zebrafish larvae whole-mount immunofluorescence was performed according to ref. 24 (link). Briefly, xenografts were permeabilized with 0.1% (w/v) Triton X-100 (Sigma) in PBS 1x and blocked with a mixture of PBS 1X, 1% BSA, 1% DMSO (Sigma), 0.05% Triton X-100, 1.5% goat serum (Dako) for 1 hour at room temperature. Primary anti-GFP rabbit polyclonal antibody Alexa 488 conjugate (Invitrogen, 1:100) and DAPI (50 μg/mL; Merck Millipore) were used and xenografts were mounted with Mowiol (Sigma). Cells seeded in glass coverslips were fixed with 4% paraformaldehyde (Thermo Fisher Scientific) followed by permeabilization with 0.5% Triton X-100 and blocking with 0.5% BSA-PBS1x. For BODIPY 493/503 costaining, 0.2 mol/L glycine (Sigma), 0.1 mg/mL saponin (Sigma), and 3% BSA was used. Primary anti-human mouse HSP60 antibody (BD Biosciences, 1:250) or mouse TOM20 (F10, Santa Cruz Biotechnology, 1:200) was used followed by incubation with secondary antibodies donkey anti-mouse Alexa 488 (Invitrogen, 1:400), donkey anti-mouse Alexa 594 (Invitrogen; 1:500), or Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific, 1:200), BODIPY 493/503 (Thermo Fisher Scientific; 0.2 μg/mL) and cells were mounted using Vectashield with DAPI (vectorlabs).