Alexa fluor 555 donkey anti rabbit igg

Coronal brain sections containing the basal temporal lobe were analyzed using immunofluorescence labeling as described previously.²² After collection, the brain tissues were fixed in 4% paraformaldehyde, subjected to standard histologic processing, and embedded in paraffin after equally cut into 4μm. The paraffin‐embedded tissues were sectioned, dewaxed, and blocked in 5% bovine serum to prevent nonspecific binding. These sections were then incubated with primary antibodies specific for ACSL4 (Abcam) and NeuN (Abcam) at 4℃ at least 8 h. After subsequent incubation with Alexa Fluor 488 donkey anti‐mouse IgG and Alexa Fluor 555 donkey anti‐rabbit IgG (Life Technologies), these sections were treated with antifading mounting medium containing 4, 6‐diamidino‐2‐phenylindole (DAPI, Southern Biotech) and coverslipped. Relative fluorescence intensity was analyzed using ImageJ software (NIH).

Core tissues that were 2 mm in diameter were obtained from the representative formalin-fixed paraffin-embedded NPC samples were sectioned at 4-µm thickness. Antigen retrieval was performed using a pressure cooker for 30 min in 0.01 mol/L citrate buffer (pH 6.0). The specimens were incubated with antibodies specific for CD163 (1:200; Abcam, catalog no. ab182422) and ISG15 (1:100; Novus, catalog no. NB600-891). Two independent pathologists (T-Z Zhang, L Zhang) who were blind to the clinical status of the patients counted the stained numbers of CD163⁺, ISG15⁺, and ISG15⁺CD163⁺ cells in the intratumoral area under a microscope. For immunofluorescence staining, the formalin-fixed paraffin-embedded specimens of NPC were incubated with antibodies specific for CD163 (1:100; Abcam, catalog no. ab182422) and ISG15 (1:100; Novus, catalog no. NB600-891), or the macrophages slide were incubated with antibodies specific for ISG15 (1:100; Novus, catalog no. NB600-891) and CD11+CD18 (1:100; Abcam, catalog no. ab13219) overnight at 4°C, followed by incubation with Alexa Fluor 555 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-mouse IgG (Life Technologies). Cells stained with the indicated antibody were imaged using a confocal laser-scanning microscope (Carl Zeiss) with a core data acquisition system.

Cells were plated on glass cover slips, grown to 80% confluence, and fixed with methanol at -20 °C for 30 min. After they were washed with cold PBS three times, the cover slips were blocked with Protein Block Serum-free Buffer (Dako, Mississauga, Ontario, Canada) for 1 h and then incubated with a rabbit anti-phospho-Cx43 antibody (1:100 diluted in Protein Block Serum-free Buffer) at 4 °C overnight. After the cover slips were washed with cold PBS three times, they were incubated with a secondary antibody (Alexa Fluor 555 donkey anti-rabbit IgG, Life Technologies) at room temperature for 1 h. After the cover slips were washed with cold PBS three times, they were counterstained with Prolong™ Gold anti-fading reagent containing DAPI (Thermo) and imaged with a Zeiss Axiophot fluorescence microscope equipped with a digital camera (Q Imaging, Burnaby, BC, Canada). Quantification of the fluorescence intensities were quantified using ImageJ 1.42 (NIH, U.S.A.). The experiments were performed in triplicate. Fluorescence intensity was calculated as fluorescence intensity of the treatment group divided by the fluorescence intensity of control group. The image background was subtracted by ImageJ software.

iPSCs collected were fixed with 4% paraformaldehyde in PBS for 15 min and washed with PBS for 3 times, 5 min each. Cells were then permeabilized and blocked with 0.1% triton X and 2.5% bovine serum albumin for 30 min and incubated with primary antibodies (mouse anti-OCT4 from Santa Cruz (cat#SC-5279), 1:250; rabbit anti-NANOG from GeneTex (cat#GTX100863), 1:400) for 30 min at 4 °C. After washed with PBS for 3 times, cells were incubated with secondary antibodies (Alexa Fluor 488 donkey anti-mouse IgG (cat#A21202), 1:500; Alexa Fluor 555 donkey anti-rabbit IgG (cat#A31572), 1:500, from Life Technologies) for 30 min at 4 °C. After washed, flow cytometry was done on LSRFortessa (BD Biosciences) and analyzed using FACSDIVA (BD Biosciences). Every sample was used for the analysis. For media change experiment, population with dual staining (both OCT4 and NANOG positive) was used to calculate the following parameters; (1) percentage of pluripotent cells in ‘Daily’ condition, (2) reduction in pluripotent cells in ‘Skip’ condition over corresponding ‘Daily’ condition. For single-cell plating experiment, population with dual staining (OCT4 and NANOG) was used to calculate the percentage of cells remaining pluripotent at day 7 after plated as single cells.

Immunofluorescence: CF488A donkey anti-chicken IgY (H+L) (Biotium, Cat no. 20166); Alexa Fluor 488 goat anti-mouse IgG (Life Technologies, Cat no. A11001); Alexa Fluor 488 donkey anti-goat IgG (Cat no. A11055); Alexa Fluor 555 donkey anti-rabbit IgG (Life Technologies, Cat no. A-31572); Alexa Fluor 546 donkey anti-mouse IgG (Thermo Fisher Scientific, Cat no. A10036); Alexa Fluor 555 goat anti-rabbit IgG (Cat no. A-21428); DyLight 649 donkey anti-mouse IgG (Jackson ImmunoResearch); Alexa Fluor 405 goat anti-rabbit IgG (Thermo Fisher Scientific, Cat no. A-31556); Alexa Fluor 647 goat anti-chicken IgG (Thermo Fisher Scientific, Cat no. A-21449); Alexa Fluor 647 donkey anti-rabbit IgG (Thermo Fisher Scientific, Cat no. A-31573); Alexa Fluor 647 donkey anti-rat IgG (Abcam, Cat no. ab150155).
Western blotting: HRP-conjugated antibody directed against the host species of the primary antibody.