Goat anti mouse igg2a

Human fetal lung fibroblasts (HFL-1; ATCC, Rockville, USA) were used between passages 17 and 20. HFL-1 (10000 cells/well) and co-cultures of HFL-1 (10,000 cells/well) and LAD2 cells (7500 cells/well), were seeded in 4-well glass chamber slides (154526; Thermo Scientific, Waltham, MA) and incubated overnight at 37 °C, 5% CO₂. The cells were then fixed in 4% paraformaldehyde for 15 min and blocked in 2% BSA-TBS containing 5% goat serum (Vector laboratories, Burlingame, CA) and 0.2% Triton-X for 30 min, followed by washing twice in tris-buffered saline (TBS). Cells were incubated for 60 min with monoclonal tryptase antibody (M7052, Dako, Glostrup, Denmark) and monoclonal PAR2 antibody (cat.nr: 35–2300, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The cells were then washed in TBS and incubated for 45 min with secondary antibodies (Thermo Fisher Scientific), goat anti-mouse IgG2a (Alexa Fluor® 647, A21241), goat anti-mouse IgG1 (Alexa Fluor® 647, A21240) or goat anti-mouse IgG1 (Alexa Fluor® 555, A21127), followed by washing in TBS. Nuclei were stained by using DAPI containing mounting medium (Dako). Cells were imaged using a VS120 slide scanner with XV image processor L100 VS-ASW (Olympus, Tokyo, Japan). Image viewer software VS-OlyVIA (version 2.9) (Olympus Soft Imaging solutions GmbH; Münster, Germany) was used for image visualization.

The viral envelop protein was detected using the pan-immune anti-flavivirus antibody (mouse IgG1/IgG2a, clone ATCC-HB-112 D1-4G2-4-15 hybridoma, ATCC, Wesel, Germany) and double stranded RNA (dsRNA) was detected with the anti-dsRNA J2 antibody (mouse IgG2a, Scicons, Budapest, Hungary). Cell markers were detected using anti-human antibodies against CX₃CL1 (rabbit polyclonal IgG, clone PA5-23062, Thermofischer Scientific, Waltham, MA), Iba-1 (rabbit polyclonal IgG, Wako, Richmond, VA) and fluorescent-labelled CX₃CR1-R-Phycoerythrin (rat IgG2b, clone 2A9-1, Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany).
The following secondary antibodies were used for staining: donkey anti-rabbit IgG fluorescent-labelled Alexa Fluor 647 (AF647) (Abcam, Cambridge, UK) and goat anti-mouse IgG2a fluorescent-labelled AF647 for flow cytometry, as well as goat anti-mouse IgG2a fluorescent-labelled AF488 and donkey anti-rabbit IgG fluorescent-labelled AF546 for confocal microscopy (all obtained from Thermofischer Scientific if not mentioned). Nuclei were stained using DAPI (Sigma-Aldrich, Saint Louis, MO). All concentrations for the use of the antibodies and stains were optimized in our laboratory.

Dissociated cells were fixed with 4% (v/v) paraformaldehyde, rinsed three times in D-PBS and blocked with 5% (v/v) horse serum (Sigma-Aldrich). CMs and hCMECs were incubated overnight at 4 °C with primary antibodies against sarcomeric α-actinin (1 : 200; Sigma-Aldrich) and CD31 (1 : 200; Thermo Fisher Scientific), respectively. The following day, the preparations were rinsed three times in D-PBS and incubated with secondary antibodies: donkey anti-mouse IgG (Stratech Scientific) or goat anti-mouse IgG^2a (Thermo Fisher Scientific) at a dilution of 1 : 200. hCFib were stained with Alexa Fluor 555-phalloidin (Thermo Fisher Scientific), a peptide that binds to and label actin in cells. All samples were then washed in D-PBS, mounted with Vectashield Antifade Mounting Medium (Vector Laboratories) and nuclei were counterstained with DAPI (1 : 1000; Sigma-Aldrich). The specimens were examined with a laser scanning confocal microscope (Leica SP8).

Immunohistochemistry was performed as described (Rich et al., 2018 (link)). Primary antibodies were used against olfactory marker protein (Omp; goat, Wako 019-22291, Research Resource Identifier [RRID]: AB_664696; 1:500), Sox10 (rabbit, gift of Vivian Lee, Medical College of Wisconsin, WI, 1:3,000; Meng, Yuan, & Lee, 2011 ; Yardley & Garcia-Castro, 2012 (link)), Tbr1 (rabbit, Abcam ab31940, RRID: AB_2200219; 1:1,000; expression data summarized in File S3) and Tubb3 (neuronal αIII tubulin; mouse IgG2a, clone TUJ1, Covance MMS-435P, RRID:AB_2313773; 1:250). Matched AlexaFluor-conjugated secondary antibodies (Molecular Probes, Thermo Fisher Scientific) were used at 1:1,000. For triple immunostaining, anti-Tubb3 was detected by a biotinylated secondary antibody (goat antimouse IgG2a, Invitrogen, 1:100), followed by Alexa350-conjugated NeutrAvidin (Molecular Probes, 1:100). Slides were mounted with Fluoromount G (Southern Biotech, Birmingham, AL) or Vectashield with 4’,6-diamidino-2’-phenylindole dihydrochloride (DAPI; Vector Labs, Burlingame, CA).