Alexa fluor 594 secondary antibody

Cell tracker Violet (Thermo fisher scientific) stained BMDCs were plated
overnight on concentrated nitric acid-treated glass coverslips. DCs were
stimulated with LPS
(100 ng ml⁻¹), pulsed with OVA
257-264 (SIINFEKL,
1 μg ml⁻¹) for
6 h, washed extensively and purified CD8 OTI T cells added for
18 h at a 1:10 or 1:2 DC:T-cell ratio before fixing in 4%
paraformaldehyde. Cells were blocked/permeabilized for 1 h
(1% serum/0.3% Triton-X), incubated for 2 h in
anti-phospho-S6 ribosomal protein^S235/236 (Cell Signalling,
dilution 1:100) washed and then with anti-rabbit Alexa Fluor 594 secondary
antibody (dilution 1:500; Invitrogen/Molecular Probes) for 1 h. The
coverslips were mounted with Hydromount (National Diagonostics) and
immunofluorescence images were captured using a Leica SP8 gate STED confocal
microscope. Images were acquired and quantified using the Leica LAX
software.

Venom glands of Lb and brains of D. melanogaster host larvae were dissected in PBS and fixed in 4% paraformaldehyde in PBS for 30 min, rinsed with PBST (PBS containing 0.1% Triton X-100 and 0.05% Tween 20), blocked with 1% bovine serum albumin in PBST, and stained overnight at 4 °C with anti-EsGAP1 primary antibody (1:500). Venom glands or brains were then washed three times in PBST and incubated with Alexa Fluor 594 secondary antibody (1:1000; Molecular Probes) for 2 h at room temperature. Samples were mounted in ProLong Gold Antifade Mountant with DAPI (Invitrogen). Fluorescence images were captured on a Zeiss LSM 800 confocal microscope and were processed using ImageJ and Photoshop (Adobe).

Amplex Red (Cat #A22177), MitoSOX Red (Cat #M36008) and Alexa Fluor 488 secondary antibody (Cat #A11008, RRID:AB_143165) were purchased from Molecular Probes (Thermofisher). N2 supplement (Cat #17502001), B27 supplement (Cat #17504044), Penicillin-Streptomycin (Cat #15140122), Laminin (Cat #23017015), Neural Induction supplement, StemPro Accutase (Cat #A11105), Geltrex (Cat #A1413301), and Calcium Green-5N (Cat #C3737) were from Gibco (Thermofisher). Dihydroethidium and Alexa Fluor 594 secondary antibody (Cat #A11032, RRID:AB_141672) were from Molecular Probes. D₁R antibody (Cat #324390) was from Millipore; D₂R antibody (Cat # sc-5303, RRID:AB_668816) was from Santa Cruz Biotechnology; Poly-l-ornithine (Cat #P4957), Dopamine (Cat #H8502), SCH 23390 (Cat #D054), Raclopride (Cat #R121), Protease Inhibitor Cocktail (Cat #P8340) and all other reagents were from Sigma-Aldrich.

Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X100. Cells were blocked with 10% goat serum for 30 min and then incubated with (SLC7A11 or GPX4) primary antibodies at 4 °C overnight. After washing in PBS, cells were incubated with Alexa Fluor 594 secondary antibody (Molecular Probes, Eugene, OR, USA) at room temperature for 1 h. Nuclei were stained with DAPI (Sigma, St. Louis, MO, USA) for 10 min. Pictures were subsequently taken on a confocal microscope or a fluorescence microscope.

Dissected tissues were fixed in 4% paraformaldehyde in HBSS, pH 7.6, at 4°C for at least 1 h, rinsed in HBSS and dehydrated in a graded ethanol series, followed by two xylene rinses, paraffin penetration and paraffin embedding. The sections were cut to 10 μm thickness by microtome and “baked” at 60°C overnight onto Superfrost/Plus Microscope glass slides (Fisher). After deparaffinizing and rinsing with PBT (phosphate-buffered saline, PBS, plus 0.1% Tween-20), the sections were placed in 90°C 0.01 M citrate buffer (pH 6.0) for 10 min, for post-fixation antigen recovery, unless otherwise noted. Sections were then treated with blocking buffer as follows: PBT with 10% normal goat serum (NGS), diluted 1:1 with Superblock (Pierce Chemical). Primary antibody was added in HBSS and incubated overnight at 4°C. After rinsing three times with HBSS, sections were incubated with Alexa Fluor 594 secondary antibody (1:2,000, Invitrogen) for 2 h at room temperature. CD36 antibody (R&D Systems MAB25191) was diluted to 2.5 ug/ml. After washing with HBSS twice, sections were mounted in SlowFade Gold antifade reagent with DAPI as the nuclear counterstain (Invitrogen) and coverslipped. Specimens were observed with a Nikon Eclipse E800 fluorescence/DIC microscope.

In vitro IEC-6 cells were post-fixed in 4% paraformaldehyde for 20 min and incubated in 0.3% Triton X-100 for 20 min. Intestinal tissue sections or cells were washed 3 times in PBS, blocked with 5% BSA solution for 1 h, and incubated with a primary antibody overnight at 4 °C. The primary antibodies were as follows: anti-rabbit β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-rabbit GSK-3β (Santa Cruz Biotechnology, Santa Cruz, CA). An Alexa Fluor 594 secondary antibody (Invitrogen Life Technologies, Carlsbad, CA, USA) was added for 1 h at 37 °C. Then, 4,6-diamidino-2- phenylindole (DAPI, Beyotime, Shanghai, China) was added as a nuclear counterstain. A Leica DM 4000B microscope or a Leica TCS SP5 microscope was used to examine staining for conventional or confocal imaging, respectively.