Alexa fluor 555 donkey anti rabbit igg antibody

Antibodies against specific molecules were obtained as follows: CD68 (catalog: ab25212, Abcam Cambridge, UK), hepatic sinusoid endothelial cells (SE-1) (catalog: NB110-68095, Novusbio, Colorado, USA), Alexa Fluor 555 donkey anti-rabbit IgG antibody (catalog: A-31572, Invitrogen), endothelial nitric oxide synthase (eNOS) (catalog: #9572, Cell Signaling, Danvers, USA), phospho-eNOS Thr495 (catalog: #9574, Cell Signaling), and horseradish peroxidase (HRP)-conjugated 2^nd Ab for WB (catalog: P0448, DAKO, Santa Clara, USA).
A polyclonal antibody against sodium-phosphate cotransporter 2a (NaPi-2a) was raised in rabbits by immunization with rat NaPi-2a peptide (MMSYSERLGGPAVSP)^{25 (link)}.

Double immunofluorescence staining was conducted as described elsewhere (27 (link)). After washing thrice with 0.3% Triton in phosphate-buffered saline (PBS) to permeabilize the cell membrane, the frozen brain sections (15 μm thick) were blocked with an immunostaining blocking buffer (Beyotime) at room temperature for 1 h and then incubated at 4°C overnight using the following primary antibodies: rabbit anti-Rab10 (phospho T73) (1:200, Abcam), rabbit anti-LRRK2 (1:200, Abcam), and mouse anti-NeuN (1:300, Cell Signaling, USA). After incubating with Alexa Fluor 488 donkey anti-mouse IgG antibody (1:800, Invitrogen) and Alexa Fluor 555 donkey anti-rabbit IgG antibody (1:800, Invitrogen) at room temperature for 1 h, the sections were stained for 10 min with 4′, 6-dididia-2-phenylindolediamine hydrochloride (DAPI), and were assessed thereafter under a fluorescence microscope (OLYMPUS, U-RFL-T, Japan).

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.

Cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS twice, permeabilized with 0.2% (w/v) Triton X-100 (Sigma) for at least one hour, washed with PBS twice and blocked with 1% BSA (sigma) for 5 hours. Alexa Fluor 555 Phalloidin (Life Technologies) was diluted at 1:30 ratio with 1% BSA solutions and applied to the fixed cells at 4°C overnight. Cells were washed three times with PBS (5 minutes each time) before imaging. For histamine H1 receptor immunofluorescence, cells were fixed and blocked as described above, and incubated overnight at 4°C with rabbit histamine H1 receptor antibody (Novus Biologicals, rabbit polyclonal IgG, 1:150 diluted). After washing three times with PBS, cells were further incubated at room temperature for 1 hour with Alexa Fluor® 555 donkey anti-rabbit IgG antibody (Life Technologies). To study the roles of calcium signaling in cytoskeletal reorganization, cells were incubated with 15 μM BAPTA-AM (EMD Millipore) for 30 minutes at 37°C in the dark and washed three times with HBSS before histamine applications. To further resolve the role of calcium oscillations, three pulses (30 seconds) of histamine solution were applied to the monolayer and single cells with an interval of 1 minute HBSS wash between pulses to mimic natural calcium oscillations.