Immunofluorescence analysis was performed according to Scardigli and collaborators 38 . Briefly, cells and cell‐laden PF constructs were fixed with 2% PFA in PBS for 30 min. at 4°C. Then, samples were washed with PBS and blocked with 10% goat serum in PBS for 1 hr at room temperature (RT). Subsequently, they were incubated with anti‐Collagen Type I primary antibody (rabbit polyclonal, #ab21286, 1:100 dilution; Abcam Cambridge, UK) followed by incubation with AlexaFluor 488 conjugated goat anti‐rabbit IgG (H+L) (Thermo Fisher Scientific #A‐11008, 1:300). Finally, nuclei were counterstained with 300 nM DAPI (Thermo Fisher Scientific MA, USA) for 10 min. Specimens were viewed under a Nikon TE 2000 epifluorescence microscope equipped with a Photometrics CoolSNAP MYO CCD camera.
Alexa fluor 488 conjugated goat anti rabbit igg h l
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Alexa Fluor 488®-conjugated goat anti-rabbit IgG (H + L) is a secondary antibody used for detection and visualization in immunoassays. It is conjugated with the fluorescent dye Alexa Fluor 488 and binds to the heavy and light chains of rabbit immunoglobulin G (IgG) antibodies.
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Lab products found in correlation
Antifade mounting medium, by Electron Microscopy Sciences (5 mentions)
Hrp labeled goat anti rabbit igg, by Cell Signaling Technology (5 mentions)
Alexa fluor 568 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
β actin rabbit mab, by Cell Signaling Technology (3 mentions)
Donkey anti goat igg h l hrp, by Abcam (3 mentions)
Alexa fluor 633 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (3 mentions)
2 well chamber slide, by Thermo Fisher Scientific (3 mentions)
Lamina c mouse mab, by Santa Cruz Biotechnology (3 mentions)
β tubulin rabbit pab, by ABclonal (3 mentions)
Alexa fluor 594conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Cell Signaling Technology (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Fv3000, by Olympus (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
8 well chamber slide, by Thermo Fisher Scientific (2 mentions)
Confocal microscope, by Leica (2 mentions)
P plc γ1 ser1248 rabbit mab, by Cell Signaling Technology (2 mentions)
Plc γ1 pab, by Cell Signaling Technology (2 mentions)
46 protocols using alexa fluor 488 conjugated goat anti rabbit igg h l
1
Immunofluorescence Analysis of Collagen Type I
2
PEDV N Protein Quantification Protocol
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The GPR43 inhibitor GLPG0974 (SML2443), acetate (S2889), propionate (P5436), and butyrate (V900464) were purchased from Sigma-Aldrich (St. Louis, MO, United States). The GPR109a inhibitor Mepenzolate bromide (MPN) (HY-17585) and the nuclear factor kappa B (NF-κB) pathway inhibitor BAY 11–7,082 (HY-13453) were obtained from MedChemExpress (Monmouth Junction, NJ, USA). The primary antibodies for western blotting were mouse monoclonal antibodies against PEDV N (Medgene Labs, Brookings, SD, USA; 1,403,113) and glyceraldehyde-3-phospahte dehydrogenase (GAPDH) (ABclonal, Wuhan, China; AC002). The secondary antibodies used for western blotting were horseradish peroxidase (HRP)-Goat Anti Mouse IgG (H + L) (EARTH OX, Millbrae, CA, United States; E030110). The Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) was purchase from Thermo Fisher Scientific (Waltham, MA, United States; A-11001).
3
Autophagy Detection in Cholangiocarcinoma
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Autophagic cells were detected with LC3 using autophagy watch (Medical & Biological Laboratories, Aichi, Japan), according to the manufacturer’s instructions. For Western blot analysis, HuCCT-1 and SSP-25 cells were treated with UA (40 μmol/L) and/or Chloroquine (CQ; 20 μmol/L) for 24 h, and the analysis was performed with 20 µL of protein lysate sample using anti-LC3 monoclonal antibody-HRP-DirecT (Autophagy watch). For immunocytochemistry, the cells were evaluated using anti-LC3 monoclonal antibody (Autophagy watch), with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L; Thermo Fisher Scientific, Waltham, MA, USA) as the secondary antibody. All sections were counterstained using 4′,6-diamidino-2-phenylindole (DAPI; Fluoromount-G; Southern Biotech, Birmingham, AL, USA). HuCCT-1 cells were seeded in 4-well glass slides (Lab-Tek® Chamber Slide™ system; Thermo Fisher Scientific) and incubated for 24 h, and then treated under the respective conditions for 24 h. Images were obtained using a confocal laser scanning fluorescence microscope (FV3000; Olympus, Tokyo, Japan).
4
Embryonic Tissue Preparation and Analysis
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Mice were euthanized at predetermined stage and embryos were collected in cold PBS, fixed in 4% PFA overnight, washed in PBS for 3 times and processed to 100% methanol (for whole mount in situ hybridization), or paraffin for section (for histology staining and section in situ hybridization). Histology staining and in situ hybridization procedures were performed as previously described (Liu et al., 2013 (link), 2015 (link)). In situ probe templates were amplified by PCR from total cDNA sample of E13.5 wildtype forelimbs. Antisense RNA probes were synthesized from templates using T7 RNA polymerase (Promega catalog# P2075). Immunofluorescent staining was performed as previously described (Xu et al., 2014 (link)). The primary antibodies used include anti-Sox9 (Santa Cruz, catalog# sc-20095, 1:25 dilution) and anti-FLAG antibody (Sigma, catalog# F1804, 1:50 dilution). The secondary antibodies were Alexa Fluor 568-conjugated goat anti-mouse IgG (H + L) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) [(Thermo Fisher Scientific, Waltham, MA, United States), catalog# A11004 and A27034, 1:500 dilution].
5
Quantification of 4-HNE in Neuronal Cultures
6
Astrocyte Characterization with Immunostaining
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Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% triton X‐100 and blocked by 1% normal goat serum, 1% BSA and 0.1% tween20 in PBS for 1 h. Those samples were then incubated at 4°C overnight with primary antibodies diluted in PBS containing 1% BSA and 0.1% tween20. Next, those samples were incubated with secondary antibodies at room temperature for 1 h. Hoechst 33342 (Doujin Chemical, Kumamoto, Japan) was used to counterstain the nuclei. The following primary and secondary antibodies were used: anti‐S100β (Abcam, Cambridge, UK, ab52642, 1:200), GFAP (Cell Signalling Technology, Beverly, MA, 3670, 1:1000), NESTIN (Millipore‐Sigma, St. Louis, MO, MAB5326, 1:500), Alexa Fluor 488‐conjugated Goat anti‐Rabbit IgG (H + L) (Thermo Fisher Scientific, A‐11034, 1:1000) and Alexa Fluor 555‐conjugated Goat anti‐Mouse IgG (H + L) (Thermo Fisher Scientific, A‐21422, 1:1000). After immunostaining steps, cells were analysed by Opera Phenix (PerkinElmer, Waltham, MA) with 10× or 40× objective lenses, followed by quantification for the positivity of astrocytic markers using Harmony software (PerkinElmer). When calculating GFAP aggregates, the images were taken using a 40× objective lens and the round areas showing strong intensity against the backgrounds were counted (Common Threshold: 0.90, Area > 10 pixels, Roundness > 0.80).
7
Immunofluorescent Analysis of DNA Damage Response
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Cells were seeded at the density of 10 × 104 cells/well onto covered slips which placed in six-well plates and incubated for 12 h. After that, the cells were exposed to 8 Gy high energy X-ray, and then put back to incubate for 24 h. Then, the cells were fixed in 4% paraformaldehyde (Solarbio) for 20 min, incubated in PBS supplemented with 0.2% Triton X-100 (Solarbio) for 10 min, and blocked in PBS supplemented with 10%FBS and 1% bovine serum albumin for 1 h at room temperature in sequence. Thereafter, the cells were incubated with rabbit monoclonal antibody gammaH2AX (γ-H2AX) (Ser139, Cell Signaling Technology, 1:400), E-cadherin (WL00941, Wanleibio, 1:200), or Vimentin (WL01960, Wanleibio, 1:200) diluted in PBS/10%FBS + 1%bovine serum albumin at 4 °C overnight. The next day, after rinsing with PBS, the cells were incubated with Alexa Fluor® 488 conjugated, goat anti-rabbit IgG (H + L) (Thermo Scientific) at 1:400 for 1 h at 37 °C in dark. Hoechst 33342 (1:10,000, Molecular Probes) was used for nuclear counterstaining. Slides were mounted using ProLong Diamond Antifade Mountant (#P36965, Invitrogen).
8
PEDV Neutralization Assay in Vero/IPEC-DQ Cells
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Vero/IPEC-DQ cell monolayers in 96-well plates were treated with 250 mU NA in DMEM/RPMI-1640 or mock DMEM/RPMI-1640 at 37℃ for 2 h. After three washings with DMEM/RPMI-1640, cells were inoculated with icPC22 A or icPC22 A-S1Δ197 at a MOI of 0.02 with 10 μg/mL trypsin for 2 h. Next, the inoculum was removed followed by three washings. DMEM/RPMI-1640 containing 50 μg/mL soybean trypsin inhibitor (SBTI) and swine PEDV positive serum (virus neutralization titer 1:1024, 1:500 dilution) was added to prevent virus spreading. Cells were fixed with acetone-methanol (1:4) at 6 hpi. The numbers of PEDV-infected cell foci were determined by IF assays as described for the staining of PEDV N proteins (Hou et al., 2017 (link)). Briefly, rabbit PAb RB9 (1:1000) was used as the primary antibody. After incubating at 4℃ overnight, cells were washed three times. Then Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) (Thermo Fisher Scientific) (1:1000) was used as the 2nd antibody and the cells were incubated at room temperature for 1 h. After washing and adding mounting medium, cells were observed and picture were captured by using Olympus IX-70 fluorescent microscope. The numbers of fluorescent focus units (FFU) were counted by ImageJ software. The percentages of FFU were calculated by the value of mock group as 100%.
9
Immunofluorescent Localization of DNA Damage Markers
10
Immunofluorescence Analysis of YAP and Survivin
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Low‐density and high‐density cultures of untreated, mock, siNC, siSurvivin‐1 and siSurvivin‐2‐treated ISO‐HAS‐B cells on 12‐well plates were fixed with 4% paraformaldehyde in 50‐mM HEPES buffer (pH 7.3) for 20 min, permeabilized for 20 min with 0.2% Triton X‐100 in TBS (pH 7.4), and treated with 5% BSA in T‐TBS for 1 h at room temperature. Cells were incubated overnight at 4°C with the primary antibodies diluted at 1:200 (anti‐YAP) and 1:400 (anti‐survivin) in T‐TBS and further incubated with secondary antibodies (Alexa Fluor 488‐conjugated goat anti‐rabbit IgG [H+L] and anti‐mouse IgG [H+L] [Thermo Fisher Scientific]) diluted at 1:100 and Alexa Fluor 594 phalloidin (Thermo Fisher Scientific) diluted at 1:40 in T‐TBS for 1 h at room temperature. Finally, cells were counterstained with Hoechst 33342 (Dojindo Laboratories) diluted at 1:100 in T‐TBS for 20 min at room temperature.
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