Alexa fluor 594 donkey anti rabbit igg

For immunocytochemistry, cells were washed twice with PBS and fixed in 4% paraformaldehyde (PFA) for 15 min, followed by permeabilization with 0.5% Triton in PBS for 5 min. Cells were then blocked with 4% BSA in PBS for one hour. After that, cells were incubated with primary antibodies in 4% BSA in PBS overnight at 4 °C. The next day, after five PBS washes, cells were incubated in secondary antibodies diluted in 4% BSA in PBS for one hour. Cells were then washed five times with PBS, and fluorescence images were acquired with a Zeiss LSM 710 confocal microscope (Zeiss International, Oberkochen, Germany). Fluorescence intensity was adjusted with ImageJ software (NIH). The primary antibodies used for immunocytochemistry were: lamin A/C (1:250, Millipore MAB3211), progerin (1:250, Cao et al., 2011 [41 (link)]), ∆Np63 (1:250, Biolegend, San Diego, CA, USA #619001), and K14 (1:500, Invitrogen, Waltham, MA, USA MA5-11599). The secondary antibodies used were: Alexa Fluor 488 donkey anti-rabbit IgG (1:1000, Invitrogen), Alexa Fluor 594 donkey anti-rabbit IgG (1:1000, Invitrogen), Alexa Fluor 488 donkey anti-mouse IgG (1:1000, Invitrogen), and Alexa Fluor 594 donkey anti-mouse IgG (1:1000, Invitrogen).

To analyze c-Fos expression evoked at 90 min after ZD7288-induced vomiting, c-Fos immunostaining was conducted on both brainstem (20 µm) and jejunal sections (25 µm) from vehicle-treated and ZD7288 (1 mg/kg, i. p.)-treated animals (n = 4 shrews per group for brainstem staining; n = 3 for jejunual sections staining). Rabbit anti-c-Fos polyclonal antibody (1:5000, ab190289, Abcam) and Alexa Fluor 594 donkey anti-rabbit secondary antibody (1:500, Abcam) were used. The criterion used to differentiate the AP, NTS and DMNX within the DVC has been well recognized (Darmani et al., 2008 (link); Ray et al., 2009a (link); Chebolu et al., 2010 (link); Zhong et al., 2016 (link); Zhong et al., 2018 (link); Zhong et al., 2019 (link); Zhong and Darmani, 2020 (link)). For each animal, c-Fos positive cells in the AP, both sides of NTS and DMNX from 3 sections at 90-μm intervals were counted manually by an experimenter blind to experimental conditions. The average value was used in statistical analysis. Co-staining of jejunum sections with anti-c-Fos antibody and anti-NeuN (neuronal marker) antibody (1:300, MAB377, Millipore) followed by Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-mouse secondary antibodies (1:500, Invitrogen) was conducted to confirm neuronal localization of c-Fos.

Frozen 5 μm-thick tissue sections were fixed in cold acetone for 15 min, air-dried, and treated for 30 min with a blocking reagent [10% fetal bovine serum (FBS) in PBS]. After washing with PBS, the sections were incubated with primary antibodies overnight at 4°C, and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) for anti-NP and anti-pSFK (Tyr416) antibodies, Alexa Fluor 594 donkey anti-goat IgG (Invitrogen) for anti-MGL1/2 antibody, Texas Red-X goat anti-rat IgG (Invitrogen) for anti-CD3 antibody, and Alexa Fluor 594 donkey anti-rabbit IgG (Invitrogen) for anti-MPO antibody for 2 hrs at room temperature (RT). The sections were mounted with CC/Mount (Diagnostic BioSystems, Pleasanton, CA) containing DAPI (Dojindo Laboratories, Kumamoto, Japan). Fluorescent images were visualized using BIOREVO BZ-9000 (Keyence, Osaka, Japan).

The immunofluorescence assay was performed according to the previously described methods [30 (link)]. The primary antibodies used were shown in Table 2. The secondary antibodies used were Alexa 488 goat-anti mouse lgG (1:1000, Invitrogen) and Alexa Fluor 594 donkey anti-rabbit IgG (1:1000, Invitrogen). The cells were analyzed under an LSM780 or LSM800 confocal microscope (Zeiss, Germany).

To visualize microglia and neurons, sections (Bregma - 1.5 mm) were incubated with rabbit anti-Iba1 (1:1000; Wako) or rabbit anti-NeuN (1:1000; Millipore) primary antibody, respectively, followed by Alexa Fluor 647 donkey anti-rabbit IgG secondary antibody (1:500, Invitrogen). In order to visualize microglial lysosomes, sections were incubated with rabbit anti-Iba1 (1:1000; Wako) and rat anti-CD68 (1:500; Abcam) primary antibodies, Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 647 donkey anti-rat IgG secondary antibodies (1:500, Invitrogen), and 0.1% Sudan Black B (Sigma-Aldrich) solution in 70% ethanol for 2 min prior to cover-slipping. Slides were then imaged using a Leica SP8 upright confocal microscope at 63X magnification and sequential optical sections captured using the Leica Application Suite X imaging software. Lipofuscin was imaged at 488 nm excitation and 495–545 nm emission. Sequential optical sections were analyzed using ImageJ software (NIH). For each animal, 2–4 images were quantified and averaged, and these values were used to calculate the mean and standard error for each experimental group.