Alexa fluor 555 goat anti mouse igg

Serum samples were obtained from blood collected from the submandibular vein at the indicated timepoints and incubated on Kallestad HEp-2 slides (Bio-Rad) according to the manufacturer’s instructions. Bound antibodies were detected using goat anti-mouse IgG Alexa Fluor 555 (Thermo Fisher Scientific). Fluorophore signals were visualized using a DMRB fluorescence microscope (Leica). We recorded the mean fluorescence intensity of 10 representative nuclei per slide using the “measure” function in ImageJ. Data are presented as the mean ± SEM for each condition.

Cells were washed once with PBS and fixed in 4% PFA for 10 min at room temperature. Cells were permeabilized (PBS + 0.5% Triton) for 5 min and blocked in blocking buffer (10% in goat serum in PBS + 5% BSA) for 1 hour at room temperature. Primary antibodies (1 : 200; Anti-α-Actinin (Sarcomeric) mouse monoclonal antibody, Sigma Aldrich and A7732) were diluted in blocking buffer containing 0.1% saponine and cells were incubated at 4°C over night. Dishes were washed twice for 5 min in PBS under agitation. Secondary antibodies (1 : 400; Goat anti-Mouse IgG Alexa Fluor 555, Thermo Fisher Scientific, A21424; Goat anti-Rabbit IgG Alexa Fluor 647, Thermo Fisher Scientific, A21245) in blocking buffer were incubated in the presence of DAPI (100 μg ml⁻¹) for 1 h. Dishes were washed twice, 200 μl Fluoromount-G (Invitrogen) was added on top of cells. Samples were stored at 4°C. Dishes were imaged with an inverted fluorescent microscope (Zeiss Cell observer) using a 40× phase air objective (EC Plan-NeoFluar NA 0.75). z-stacks of 1 μm were taken as 3 × 3 tiles with 10% overlap and 1 × 1 binning. Subsequently, images were stitched in Zen Blue.

Double fluorescent immunolabelings were carried out to determine the co-localization of IL-1β and NLRP proteins with other markers. Before antibody treatments, tissue sections were kept in 10% normal goat serum (Vector Labs) dissolved in PBS for 50 min, then incubated with a selection of several antibodies that contained either (a) rabbit anti-IL-1β, (b) rabbit anti-NLRP1, (c) rabbit anti-NLRP2 (d) rabbit anti-NLRP3 and one of the following antibodies: (e) mouse anti-glial fibrillary acidic protein (GFAP) (diluted 1:500; Chemicon, Temecula, CA, USA; catalog no. MAB3402), (f) guinea-pig anti Iba1 (diluted 1:2000; Synaptic Systems, Goettingen, Germany; catalog No. 234-004). Sections were gently shaken in the primary antibody solutions for 2 days at 4 °C and were further placed into the proper combination of secondary antibodies for 2 h selected from the following: (a) goat anti-rabbit IgG conjugated with Alexa Fluor 488 (diluted 1:1000; Thermo Fisher Scientific, Waltham, MA, USA; catalog No. A11034), (b) goat anti-mouse IgG-Alexa Fluor 555 (diluted 1:1000, Thermo Fisher Scientific, catalog no. A21422), (c) goat anti-guinea-pig IgG-Alexa Fluor 555 (diluted 1:1000, Thermo Fisher Scientific, catalog No. A21435). Sections were covered with mounting medium and Vectashield (Vector Labs) on glass slides.

For confocal microscopy, cells were fixed in 4% paraformaldehyde after treatments, permeabilized with 0.3% Triton X-100, blocked with 5% bovine serum albumin, and then incubated with primary antibodies overnight at 4°C. Next, the cells were incubated with fluorescent dye-labeled secondary antibodies: Donkey Anti-Rat IgG Alexa Fluor 594 (1:1,000, A-21209, Thermo Fisher Scientific), Donkey Anti-Rabbit IgG Alexa Fluor 555 (1:1,000, A-31572, Life Technologies), Goat Anti-Rabbit IgG Alexa Fluor 488 (1:1,000, A-11008, Life Technologies), Goat Anti-Mouse IgG Alexa Fluor 488 (1:1,000, A-11001, Thermo Fisher Scientific), and Goat Anti-Mouse IgG Alexa Fluor 555(1:1,000, A-21422, Thermo Fisher Scientific). Nuclei were stained with DAPI. Confocal fluorescence images were captured using a Nikon confocal microscope.

Male and female FVB/nj:C57BL/6j Ercc1^–/– and their respective control WT mice^{81 (link)}, on the third day after birth (postnatal day P3), were injected intraperitoneally 3 times per week with 10 mg/kg body weight of harmine hydrochloride (SMB00461, Sigma) diluted in 0.9% sodium chloride. Mice were euthanized at postnatal day P15 for retina tissue isolation. Tissues were embedded in optimal cutting temperature (OCT) compound, cryosectioned and stained using the in situ cell death detection kit (TUNEL staining) (11684817910, Roche), according to the manufacturer’s instructions.
For the immunostaining experiments against γH2AX (Millipore, 05–636), retina slices were fixed in 4% formaldehyde in 1× PBS for 10 min at room temperature, permeabilized with 0.5% Triton X-100 in 1× PBS for 10 min, on ice, and blocked with 1% BSA in 1× PBS for 1.5 h at room temperature. After overnight incubation with the primary antibody (1:12,000, in 1% BSA/1× PBS, 4 ^oC), a secondary fluorescent antibody was added (goat anti-mouse IgG-Alexa Fluor 555, 1:2,000, Invitrogen, A-21422) and DAPI (1:20,000, Thermo Fisher Scientific, 62247) was used for nuclear counterstaining.
Samples were visualized with an SP8 TCS laser scanning confocal microscope (Leica). The detection of nuclei and signal intensity from retinas was performed utilizing Imaris 9.9 (Oxford Instruments).

Anti-SYCP3 (1:5000, [26 (link)]), previously generated rabbit anti-HORMAD1 (1:200, [27 (link)]), previously generated rabbit anti-RPA (1:50, [28 (link)]), rabbit polyclonal anti-H3S10p (1:1000, Upstate, 06-570), mouse anti-MLH1 (1:25, BD Pharmingen, 51-1327GR), mouse monoclonal anti-γH2AX (1:10,000, Upstate, 05-636), and mouse monoclonal anti-SYCP3 (1:200, Abcam, ab97672) were used. The following secondary antibodies were used: goat anti-rabbit IgG Alexa Fluor 488 (1:500, Invitrogen A-11008), goat anti-rabbit IgG Alexa Fluor 546 (1:500, Invitrogen A-11010), goat anti-mouse IgG Alexa Fluor 488 (1:500, Invitrogen A-11001), and goat anti-mouse IgG Alexa Fluor 555 (1:500, Invitrogen A-21422).