Alexa fluor 488 donkey anti rabbit igg antibody

Notch1 antibody (ab27526), Chac1 antibody (ab76386), Rb pAb to activated Notch1 (ab52301), Rb mAb to NeuN (ab177487), Ms mAb to NeuN (ab104224) were from Abcam. β-tubulin (sc-9014), TGN 38 (sc-27680), normal rabbit IgG (sc-2027), normal mouse IgG (sc-2025) and normal goat IgG (sc-2028) were from Santa Cruz Biotechnology. Anti-Botch1/Chac1 (75-181) was from Neuromab. Cleaved caspase-3 (D175) was from Cell Signaling Technology. Protein A+G Agarose (P2012) was from Beyotime. Secondary antibodies for western blot analysis, including goat anti-rabbit IgG-HRP (sc-2004), donkey anti-goat IgG-HRP (sc-2020), and goat anti-mouse IgG-HRP(sc-2005) were from Santa Cruz Biotechnology. Secondary antibodies for immunofluorescence, including Alexa Fluor-488 donkey anti-rabbit IgG antibody (A21206), Alexa Fluor-488 donkey anti-mouse IgG antibody (A21202), Alexa Fluor-555 donkey anti-mouse IgG antibody (A31570), Alexa Fluor-555 donkey anti-rabbit IgG antibody (A31572), and Alexa Fluor-633 donkey anti-goat IgG antibody (A21082) were from life technologies.

We performed double labeling for TDP-43 and NeuN to assess expression of TDP-43 in neurons. The rat brain samples were fixed in 4% paraformaldehyde and embedded in paraffin. Next, sections were incubated with the primary antibody (TDP-43, 1:100) overnight at 4°C followed by incubation with the NeuN antibody (neuronal cell marker, 1:100) overnight at 4°C. Then, sections were incubated with the secondary antibodies, which included Alexa Fluor 488 donkey anti-rabbit IgG antibody, Alexa Fluor 555 donkey anti-mouse IgG antibody, Alexa Fluor 488 donkey anti-mouse IgG antibody, and Alexa Fluor 555 donkey anti-rabbit IgG antibody (Life Technologies, Carlsbad, CA, USA, 1:300). Normal rabbit IgG and normal mouse IgG were used as negative controls (data not shown). Finally, sections were observed using a fluorescence microscope (Olympus BX50/BX-FLA/DP70, Olympus Co., Japan), and relative fluorescence intensity was analyzed using ImageJ software.

hCMEC/D3 were grown to confluence on glass coverslips (Thermo Scientific) coated with rat tail collagen type I solution and then washed in PBS and fixed with methanol 15 minutes at RT (for tight junctions) or in PFA 4% 30 minutes in 4 °C (for LRP1). Fixed cells were permeabilized with 0.1% TritonX-100 and blocked with 5% BSA followed by incubation with primary antibodies against claudin-5 (Rabbit anti Claudin5, Abcam, 1:200), occludin (Rabbit anti Occludin, Invitrogen™, 1:50) or LRP1 (Rabbit anti LRP1 antibody, Abcam 1:100). After washing, cells were incubated with Alexa Fluor-488 donkey anti-rabbit IgG antibody (Life technologies, 1:1000). Then, coverslips were mounted with Fluoroshield™ with DAPI (Sigma-Aldrich) and visualized and photographed with Confocal Microscope (Leica SP2 AOBS SE) for tight junctions or by Zeiss Axio Imager Z1 equipped with an Axiocam MR3.0 camera and Axiovision 4.7 software.

The whole skin, containing epidermis and dermis, was post-fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). The samples were embedded, and the frozen blocks were cut using a cryostat (Leica Microsystems, Tokyo, Japan). The sections were reacted with a rabbit anti-rat AQP3 antibody (Alomone Labs, Jerusalem, Israel). The sections were treated with an Alexa Fluor 488 donkey anti-rabbit IgG antibody (Invitrogen Corp., Tokyo, Japan) and a 4′,6-diamidino-2-phenylindole solution (Wako Pure Chemicals, Osaka, Japan). The immunostained sections were observed under a fluorescent microscope (FV1200 IX83, Olympus Corporation, Tokyo, Japan).

Eyeball sections were deparaffinized and permeabilized using 0.05% Triton X-100. The samples was blocked by 3% bovine serum albumin and incubated with primary antibodies against caspase 3 (1:50), caspase 9 (1:100) (Proteintech Group, USA) overnight at 4 °C and washed with PBST. Sections were further incubated with anti-rabbit secondary antibody (1:500) (Jackson ImmunoResearch Inc., USA) followed by peroxidase staining with the peroxidase substrate 3,3-diaminobenzidine tetrahydrochloride. Sections were counterstained, mounted, and observed under a microscope.
For immunofluorescence staining of retinal cross sections, tissues were treated in 1% goat serum albumin/PBS for 1 h at room temperature before incubation with the primary antibody against PARP1 (1:200; Proteintech Group, USA) overnight at 4 °C. After several washes, sections were incubated with the secondary antibody Alexa Fluor 488 donkey anti-rabbit IgG antibody (1:100, Invitrogen, USA) at room temperature for 1 h and washed with PBS. The sections were counterstained with DAPI (Invitrogen, USA) in PBS. The immunostaining results were analyzed by Image-Pro plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Selected embryos were fixed in 4% paraformaldehyde for 1 h at 4°C, washed in PBS, and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. After washing in PBS, embryos were incubated for 1 h in blocking solution (Novocastra Protein Block, ref^a RE7102, Novocastra Laboratories Ltd., Newcastle upon Tyne, UK). This blocking step was followed by overnight incubation at 4°C with the following antibodies (Santa Cruz Biotechnology), diluted 1 : 100 in blocking solution: rabbit polyclonal anti-ATX (sc-66813), PLA2 (sc438), LPAR1 (sc-22207), LPAR2 (sc-25490), LPAR3 (sc25492), and LPAR4 (sc-46021). Antibodies' specificity in bovine tissue was previously confirmed, through WB and IHC [12 (link), 21 (link)]. Negative control sections were incubated with normal rabbit irrelevant IgG (concentration 1 : 100, sc-2027). After washing in PBS, embryos were incubated for 1 h at room temperature with Alexa Fluor 488 donkey anti-rabbit IgG antibody (concentration 1 : 300 in blocking solution, A-21206, Invitrogen, Life Technologies, Foster City, CA, USA). Following a final washing in PBS, embryos were mounted in Vectashield with DAPI (ref. H-1200, Vector Laboratories, Brunschwig Chemie, Amsterdam, Netherlands) and examined under a Leica DMRA fluorescence microscope.