Alexa fluor 568 conjugated goat anti rat igg

Mice received 1 μL ITV injection of either vehicle or 3K3A-APC, and one hour later LPS was injected ITV. At 24 h post-EIU, mice were sacrificed and eyes were removed, punched with a 30 g needle and fixed in 4% PFA for 2 h at RT. Eyes were washed with increasing concentrations of sucrose in PBS and incubated with a final concentration of 30% sucrose overnight at 4 °C. Eyes were then embedded in OCT compound (Sakura Finetek, Tokyo, Japan) on dry ice and kept at −80 °C. Serial sections of 10 µm thickness were cut using a cryostat (Leica Biosystems, Wetzlar, Germany). Sequential cryosections of every fifth section of each eye were blocked with 10% NDS for 1 h at RT and then incubated with rat anti-mouse CD11b (1:200, Abcam, Cambridge, UK), rabbit anti-mouse Iba1 (WAKO, Hiroshima, Japan) (2 µg), rabbit anti-human IL1β (1:25, Abcam, Cambridge, UK) or rabbit anti-mouse NLRP3 (1:200, Abcam, Cambridge, UK) at 4 °C overnight. The next day, sections were incubated with Alexa Fluor 568 conjugated goat anti-rat IgG or Alexa Fluor 488 conjugated donkey anti-rabbit secondary antibody (1:100, Invitrogen, Waltham, MA, USA). Nuclei were counterstained with DAPI (Nucblue fixed cell stain, Molecular Probes, Eugene, OR, USA). Images were captured using a fluorescence microscope (Axio Imager.Z2, Carl Zeiss Microscopy GmbH, Oberkochen, Germany).

For immunolabeling, AX2 wild-type or ndrC-null cells, settled onto glass coverslips, were fixed with a mixture of 15% (v/v) of saturated picric acid and 2% (w/v) paraformaldehyde in 10 mM Pipes-HCl, pH 6.0, at room temperature for 20 min, and post-fixed with 70% ethanol for 10 min. α-tubulin was detected using monoclonal rat antibodies (YL1/2) [43 (link)] and Alexa Fluor 568-conjugated goat anti-rat IgG (Invitrogen). For visualization of filamentous actin, cells were stained with Alexa Fluor 488-phalloidin (Molecular Probes). DNA was visualized either with TO-PRO-3 or with DAPI.
Polyclonal antibodies were generated against GST-tagged NdrC-RBD. The sequence encoding amino acid residues 1 to 299 of NdrC was cloned into the bacterial expression vector pGEX-6P1 (GE Healthcare). Purification of the GST-fusion protein was performed using standard procedures, and the recombinant protein was used to immunize a female white New Zealand rabbit together with the adjuvant Gerbu100 (Gerbu Biochemicals).

IgA and Gd-IgA1 deposition in allograft biopsy specimens and tonsil tissues was examined using immunofluorescence staining. Paraffin-embedded sections of 4-μm thickness were prepared for staining. After deparaffinisation in a PathoClean®/ethanol series and rehydration, antigen retrieval with subtilisin A (P5380, Sigma-Aldrich) was performed at room temperature for 2 h. Next, samples were blocked with non-fat dry milk (#9999S, Cell Signaling Technology) at room temperature for 60 min, followed by incubation with KM55 (1:10 dilution; #10777, Immuno-Biological Laboratories) at 4°C overnight. After several washes with phosphate-buffered saline, an Alexa Fluor 568-conjugated goat anti-rat IgG (1:200 dilution; Life Technologies) was added at room temperature for 60 min followed by incubation with fluorescein isothiocyanate-conjugated polyclonal rabbit anti-human IgA (1:50 dilution; Dako) at 37°C for 30 min. Fluorescence was observed using a BZ-X800 microscope (Keyence).