Anti mouse alexa fluor 488 conjugate

For immunofluorescence analysis, cells were plated in 6-well plates containing coverslip. After treatment (if present), cells were processed as follows: fixation in 4% formaldehyde/PBS (15 min at room temperature, RT), permeabilization in 0.5% v/v Triton X-100/PBS (10 min at RT) and blocking in 5% v/v goat serum and 0.1% v/v Triton X-100/PBS (1 h at RT). Immunofluorescence staining was performed with overnight incubation of the following antibodies: anti-STAT1 (1:200, Cell Signaling Technology), anti-STAT3 (1:1000, 124H6, Cell Signaling) and anti-Cleaved Caspase-3 (1:400, 5A1E, Cell Signaling Technology). Then, cells were washed twice in PBS and incubated with secondary antibody anti-mouse Alexa Fluor-488 conjugate or anti-rabbit Alexa Flu-or-488 or Alexa Fluor-567 conjugate (1:200) (Thermo Fisher Scientific) for 1 h at RT in the dark. An antifade mounting reagent containing DAPI was used to mount the coverslip onto the slide. Slides were observed under a fluorescence microscope (Carl Zeiss microscopy Gmbh, Jena, Germany) using a 20× or 40× objective.

Twenty-four hours after transfection, siC and siKFL7 OV-90 and PEO1 cells were seeded in 4-well chamber slides in complete growth medium and incubated for further 48hs. Thereafter, cells were fixed in 4% paraformaldehyde for 20 min at 20 °C and then blocked with 5% v/v goat serum in PBS for 1 h. Immunofluorescence staining was obtained using anti-CD44 (M7082, Dako Agilent Technologies, 1:200), following overnight incubation at + 4 °C. After washing, cells were incubated with secondary antibody anti-mouse Alexa Fluor-488 conjugate (1:200) (Thermo Fisher Scientific, Walthman, MA, USA), in the dark for 30 min at 20 °C. Coverslip was mounted onto slides using an antifade mounting reagent containing DAPI. Slides were observed under a fluorescence microscope (Leica Biosystems, Newcastle, UK) using a 40X objective. Appropriate controls were included to evaluate non-specific staining of the secondary antibody or background autofluorescence.

Transverse cryosections (14 µm thickness) were cut onto Superfrost Plus slides (Thermo Scientific, 10149870) as described by Rayon et al. (2020) (link). Slides were stored at −80°C until ready to be processed. The RNAscope multiplex fluorescent v2 kit was used as per the manufacturer's instructions for fixed frozen samples (ACD Bio), with the protease treatment step shortened to 15 min. The RNAscope probe Hs-NKX6-2 (48674) was used. At the end of the RNAscope protocol, sections were fixed in 4% paraformaldehyde for 15 min at room temperature and then washed twice in 1× PBS for 5 min. Sections were incubated in blocking solution (1% bovine serum albumin, 0.1% Triton X-100 in 1× PBS) for 30 min at room temperature and then incubated in primary antibody (DHSB, F55A10, 1:100) in blocking solution 1 h at 4°C. Sections were then washed three times for 5 min each in 1× PBS, incubated with secondary antibody (donkey anti-mouse Alexa Fluor 488 conjugate; Invitrogen, A32766, 1:1000) for 30 min at room temperature, rinsed in 1× PBS three times for 5 min each, and mounted with ProLong Gold with DAPI mounting medium (Thermo Fisher Scientific). Sections were imaged using a 40× oil immersion lens on a Leica SP8 confocal microscope. Images were taken with the pinhole set to 2 AU. Three sections of a single human embryo were recorded per stage.

Third-instar larval imaginal eye discs and adult eyes were dissected in PBS, fixed in 4% (w/v) paraformaldehyde (PFA) in 0.05% (v/v) TX-100 in PBS for 20 min at RT then permeabilised for 20 min at RT in 0.5% (v/v) TX-100/PBS, before blocking for 30 min in 5% (w/v) BSA in 0.05% TX-100/PBS. Subsequently, fixed and permeabilised discs were incubated overnight with rat anti-HA-biotin high affinity antibody (Clone 3F10, Roche) and mouse anti-CLU antibody (G7), as above. Samples were then incubated overnight in streptavidin Alexa Fluor 594 conjugate (Invitrogen; 1:10,000) and anti-mouse Alexa Fluor 488 conjugate (Invitrogen; 1:1000). All antibodies were diluted in the blocking buffer described above. Tissue was counterstained with TOTO-3 (Invitrogen; 1:10,000) diluted in 0.05% TX-100/PBS to detect nucleic acids.

Slides were fixed with INKV antigen, a positive fluorescence signal from 40–50% of the cells was required for a positive result. The samples evaluated as borderline had a positive fluorescence signal from less than 40% of the cells [33 (link)]. Two INKV positive controls; anti-Tahyna mouse-ascites and INKV positive deer whole blood were included in each run.
Initially, mouse ascites (1:100), deer whole blood, and reindeer serum samples (1:20), were diluted in 1X PBS (Dulbecco’s solution A, pH = 7.4, Norwegian Institute of Public Health, Oslo, Norway).
Twenty μl of the diluted reindeer sera (n = 480) and the controls were analysed on slides and incubated at 37 °C for 1 h, and washed with cold PBS and water. The slides were stained with anti-mouse Alexa Fluor® 488 conjugate (Invitrogen Life Technology, Inc., Carlsbad, CA, USA) (dilution1:30) and anti-deer IgG fluorescein isothiocyanate conjugate (FITC) (KPL, Gaithersburg, MD, USA) (dilution 1:25) for 30 min at 37 °C and washed as described previously. Each slide was air dried, coated with glycerol before examining with a fluorescence microscope (Nikon Eclipse Ci, Nikon Corporation, Tokyo, Japan). The overall seroprevalence of IgG against INKV in reindeer sera was calculated as the percentage of the total number of positives of the total number of samples tested.