Donkey anti rabbit igg

Anesthetized mice were perfused transcardially with heparinized PBS followed by 10% NBF. Three-mm pieces were coronally using a cutting matrix and immersed overnight in 10% NBF and then embedded in paraffin. Ten μm FFPE brain sections were deparaffinized and rehydrated. Antigen retrieval was then performed using 10 mM sodium citrate buffer (pH 6.0). Brain sections were stained with anti-NeuN (Clone A60; Millipore, Darmstadt, Germany) or anti-APC (Clone CC-1; Abcam, Cambridge, UK) for 30 min at room temperature using the Mouse-On-Mouse Fluorescein Kit (Vector, Burlingame, CA), then stained overnight at 4°C with rabbit anti-VP1 [graciously provided by R. Garcea (University of Colorado, Boulder, CO)] followed by donkey anti-rabbit IgG conjugated to Alex Fluor 594 (Jackson ImmunoResearch, West Grove, PA). Astrocyte staining was performed using directly conjugated anti-GFAP (Clone GA5; eBioscience, San Diego, CA). Tissue sections were then mounted with ProLong Gold Anti-Fade Reagent with DAPI (Life Technologies, Carlsbad, CA). Images were acquired using a Leica DM4000 B LED microscope (Leica-Camera, Wetzlar, Germany).

Western blot analysis was performed largely as described [53 (link)]. Animal caps (10–15 per condition) were lysed in 5ul/animal cap of lysis buffer (150 mM NaCl, 20 mM Tris pH 7.5, 1% Nonidet P-40, 1 mM EDTA, and 1 protease and phosphatase inhibitor tablet/10 ml of buffer) (Thermofisher) [53 (link)]. After incubation on ice for 30 min, animal cap lysates were centrifuged at 4 °C for 5 min at 14,000 g. Clear supernatant was retained. 5–15 ul of supernatant was run for each sample. Antibodies against phospho-Smad1/5 (S463/465) (Cell Signaling Technology), Smad1 (Cell Signaling Technology), Phospho-Smad2 (S465/467) (Cell signaling technology), Smad2/3 (Cell signaling technology) and GAPDH (Sigma) were all used at 1:1000 dilution. Secondary antibodies (donkey anti-rabbit IgG, or donkey anti-mouse IgG) coupled to horseradish peroxidase (Jackson ImmunoResearch) were used at 1:10,000 dilution. Bands were subjected to densitometric analysis and graphed.

The cells were seeded onto coverslips in 4-well plates. After 24 h, the cells were treated with either LPS, IRF1 siRNA, or IFITM1 siRNA; washed with PBS; and then fixed with 4% paraformaldehyde. Next, the cells were treated with cold methanol for 5 min and blocking solution (5% BSA in PBS) for 1 h. The cells were then incubated with the primary antibody anti-rabbit IFITM1 (1:200, Abcam, Cambridge, UK) and the secondary antibody donkey anti-rabbit IgG (Jackson Laboratory, West Grove, PA). Thereafter, the cells were washed with PBS, mounted with a 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution (Vectashield, Vector Laboratories, Burlingame, CA), and imaged by microscopy (Nikon Eclipse 80i Tokyo, Japan).

Eye-antennal discs from wandering third instar larvae were dissected, and fixed in 4% paraformaldehyde in Phosphate Buffered Saline (PBS), and stained following the protocol^{85 (link)–87 (link)}. The primary antibodies used were rabbit anti-Dlg (1:200; a gift from K. Cho), mouse anti-Wg [1:50,Developmental Studies Hybridoma Bank,(DSHB)], rat anti-Elav (1:50; DSHB), mouse anti-Dlg (1:100; DSHB), mouse anti-22C10 (1:100; DSHB), mouse anti-Chaoptin (MAb24B10) (1:100; DSHB⁸⁸ ), mouse anti-Ey (1:100, DSHB), mouse anti-Eya (1:100, DSHB), mouse anti-Dac (1:100, DSHB), mouse anti-β-galactosidase (1:100; DSHB), rabbit anti-β-galactosidase (1:200) (Cappel), and mouse anti-GFP (1:100, GFP-G1, DSHB). Secondary antibodies (Jackson Laboratories) used consisted of donkey anti-rabbit IgG conjugated with FITC (1:200), donkey anti-mouse IgG conjugated with Cy3 (1:250), and goat anti-rat IgG conjugated with Cy5 (1:250). The tissues were mounted in Vectashield (Vector labs) and all immunofluorescence images were captured using the Laser Scanning Confocal Microscopy⁸⁹ (Olympus Fluoview 1000). All images were taken at 20X magnification unless stated otherwise. The final images and figures were prepared using Adobe Photoshop CS6 software.

Imaginal discs were dissected from first-, second-, and third-instar larvae in 1XPBS (Phosphate Buffered Saline) and were fixed in 4% para-formaldehyde for 20 minutes. Imaginal discs were washed in PBS after fixation and stained following the standard protocol [47 (link)–49 (link)]. Antibodies used were mouse anti-Wg (1:50) (Developmental Studies Hybridoma Bank), mouse anti-Dlg (1:100), mouse anti β-gal (1:100), rabbit anti-Dlg (1:200; a gift from K. Cho), rat anti Elav (1:100). Secondary antibodies (Jackson Laboratories) used in this study were goat anti-rat IgG conjugated with Cy5 (1:250), donkey anti-rabbit IgG conjugated to Cy3 (1:250), donkey anti-mouse IgG conjugated to FITC (1:300), and donkey anti-mouse IgG conjugated to Cy3 (1:200). The imaginal discs were mounted on slides in Vectashield mountant (Vector Laboratories). Immunofluorescent images were obtained using the Olympus Fluoview 1000 Laser Scanning Confocal Microscope[50 ]. The confocal images were processed using the Photoshop CS6 software.

Cells were permeabilized with 0.2% Triton X-100 (t-octylphenoxypolyethoxyethanol, Sigma-Aldrich) in DPBS for 10 min and rinsed with DPBS. Cells were blocked with 5% normal donkey serum (EMD Millipore)/0.02% (v/v) Triton X-100 in DPBS for 4 h at room temperature or overnight at 4 °C. Primary antibodies [rabbit anti-p53 (7F5) (Cell Signaling 2527S, Lot 8), mouse anti-Mdm2 (2A10) (Abcam ab16895, Lot GR324625–5)] were applied at 1:1000 and 1:200 dilutions, respectively, in blocking buffer and incubated overnight at 4 °C. Cells were washed with DPBS six times for 5 min each. Secondary antibodies [donkey anti-rabbit IgG (Jackson ImmunoResearch) and donkey anti-mouse IgG (Jackson ImmunoResearch)] were labeled as previously described with ATTO 488 (ThermoFisher Scientific) or Alexa Fluor 647 (ThermoFisher Scientific) for a dye ratio of ∼1:1 (51 (link)). Secondary antibodies were added at 3 μg/ml each in blocking buffer and incubated for 2 h at room temperature in the dark. All subsequent steps were performed in the dark. Cells were washed with DPBS six times for 5 min each. Antibody stacks were crosslinked by 4% (v/v) paraformaldehyde in DPBS for 15 min. Remaining fixative was quenched and washed with DPBS-G twice for 5 min each, followed by DPBS twice for 5 min each. Stained cells were stored at 4 °C for up to 2 weeks before imaging.