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Immunofluorescence microscopy

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Immunofluorescence microscopy is a technique used to visualize and analyze the distribution and localization of specific proteins or molecules within cells or tissues. It involves the use of fluorescent-labeled antibodies that bind to target proteins, allowing their detection and visualization under a specialized microscope.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions) Goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions) Image pro plus 6, by Media Cybernetics (1 mentions) Dcfh da, by Beyotime (1 mentions) Click it edu imaging cell proliferation assay, by Thermo Fisher Scientific (1 mentions) Vectashield, by Vector Laboratories (1 mentions) Ab28364, by Abcam (1 mentions) Ab5694, by Abcam (1 mentions) Jc 1 dye, by Thermo Fisher Scientific (1 mentions) Facscanto 2 cell analyzer, by BD (1 mentions) Dab detection kit, by Wuhan Servicebio Technology (1 mentions) 12 well plate, by Corning (1 mentions) Cytoflex, by Beckman Coulter (1 mentions)

11 protocols using immunofluorescence microscopy

1

Transwell Invasion and Migration Assay

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A 24-well cell culture Transwell chamber (Corning Costar, San Diego, CA, USA) was used to analyze the cell invasion capacity. The filters of the upper inserts were coated with Matrigel before being used. The upper inserts were seeded with 1x10 5 cells/200 µl in RPMI-1640 medium and the lower inserts were filled with RPMI-1640 medium, supplemented with 20% FBS. After stimulation with different nucleotides (ATP, BzATP or ADP), the cells that invaded through the Matrigel and filters were fixed with methanol, and the nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI). Nuclei were counted using immunofluorescence microscopy (Nikon, Tokyo, Japan) at x200 magnification.
Transwell migration assay. Cell migration capacity was also determined using 24-well cell culture Transwell chambers (Corning Costar). Briefly, the upper inserts were seeded with 5x10 5 cells/200 µl in RPMI-1640 medium, and the lower inserts were filled with RPMI-1640 medium supplemented with 20% FBS as a chemoattractant. The cells were stimulated with or without the different nucleotides (ATP, BzATP or ADP). After an 18-h incubation at 37˚C, the migrated cells were fixed with methanol, and the nuclei were labeled with DAPI. Finally, the nuclei were counted in seven random fields using immunofluorescence microscopy (Nikon) at x200 magnification.
Xia J., Yu X., Tang L., Li G, & He T. (2015). P2X7 receptor stimulates breast cancer cell invasion and migration via the AKT pathway. Oncology reports, 34(1).
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2

Immunofluorescence Quantification of Cx43

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Rat heart samples were fixed in 4% paraformaldehyde and embedded in paraffin. Heart tissues were cut into 4–5 μm sections for immunofluorescence detection of Cx43. The polyclonal rabbit anti-Cx43 antibody (Millipore) was used at a dilution of 1:100 as the primary antibody and goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:200 as the secondary antibody. The cardiomyocytes were counterstained with DAPI (Beyotime, Jiangsu, China). Immunofluorescence was detected by immunofluorescence microscopy (Nikon, Tokyo, Japan). Cx-43 density measurements were analyzed by Image J software. Cx-43 remodeling was defined by the percentage of Cx-43 at the intercalated disks.
Zhou P., Yang X., Yang D., Jiang X., Wang W.E., Yue R, & Fang Y. (2020). Integrin-Linked Kinase Activation Prevents Ventricular Arrhythmias Induced by Ischemia/Reperfusion Via Inhibition of Connexin 43 Remodeling. Journal of Cardiovascular Translational Research, 14(4), 610-618.
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3

Cellular Proliferation Assay Using EdU

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Cells (1 × 103 cells/well) were seeded into a 24-well plate with a chamber slide. MLD was added to the cells and incubated for 24 h. Cells were fixed using 4% formaldehyde, washed with PBS, and permeabilized with 0.5% triton for 10 min. After washing with PBS, the cells were stained with Edu and DAPI, and the images were observed using immunofluorescence microscopy (Nikon).
Dai X., Sun F., Deng K., Lin G., Yin W., Chen H., Yang D., Liu K., Zhang Y, & Huang L. (2022). Mallotucin D, a Clerodane Diterpenoid from Croton crassifolius, Suppresses HepG2 Cell Growth via Inducing Autophagic Cell Death and Pyroptosis. International Journal of Molecular Sciences, 23(22), 14217.
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4

Histological Quantification of Kidney Injury

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Quantitative scoring of tubular injury was assessed on PAS-stained sections using a semiquantitative scale as described previously.40 (link) Tubulointerstitial fibrosis was assessed on Masson stained sections as the estimated percentage of blue area to total area.41 (link) Frozen OCT fixed kidney tissue sections (5 μm) slides were incubated with antibodies of α-SMA, FN, COL-1, SOCS1, CD68, and CD3 were incubated at 4°C overnight followed by incubation with secondary antibodies conjugated with Alexa 568 or Alexa 488 (Table S2) at room temperature for 1 h. After three washes with PBS, slides were mounted with 4’, 6-diamidino-2-phenylindole (DAPI) medium for 10 min. Images were captured using immunofluorescence microscopy (Nikon, Tokyo, Japan). Staining intensity was semi-quantified by Image-Pro Plus 6.0 (Media Cybernetics, MD, USA) as previously described.25 (link),26 (link)
Luan J., Fu J., Wang D., Jiao C., Cui X., Chen C., Liu D., Zhang Y., Wang Y., Yuen P.S., Kopp J.B., Pi J, & Zhou H. (2020). miR-150-Based RNA Interference Attenuates Tubulointerstitial Fibrosis through the SOCS1/JAK/STAT Pathway In Vivo and In Vitro. Molecular Therapy. Nucleic Acids, 22, 871-884.
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5

Measurement of Cellular ROS Levels

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Treated cells were washed with PBS and incubated with serum‐free basal medium, which contained 2,7‐dichlorodihydrofluorescein diacetate (DCFH‐DA, Beyotime), for 20–30 min at 37°C in the dark. The cells were washed with serum‐free cell culture solution, and then, the fluorescence intensity in the cells, which represented ROS production, was measured by immunofluorescence microscopy (Nikon Corporation, Tokyo, Japan) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
Deng X., Liang X., Yang H., Huang Z., Huang X., Liang C., Kuang Y., Qin Y., Lin F, & Luo Z. (2021). Nicotinamide mononucleotide (NMN) protects bEnd.3 cells against H2O2‐induced damage via NAMPT and the NF‐κB p65 signalling pathway. FEBS Open Bio, 11(3), 866-879.
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6

Immunofluorescence Assay for Protein Detection

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Cells (1 × 103 cells/well) were seeded into a 24-well plate with a chamber slide. MLD was added to the cells and incubated for 24 h. Cells were fixed using 4% formaldehyde for 15 min, permeabilized with 0.5% triton for 10 min, blocked with 5% BSA for 1 h, and incubated with primary antibodies overnight at 4 °C, and specific proteins were detected using secondary antibodies. After DAPI staining was performed, images were observed using immunofluorescence microscopy (Nikon, Tokyo, Japan). The signal intensities in the samples under different conditions were analyzed by using ImageJ.
Dai X., Sun F., Deng K., Lin G., Yin W., Chen H., Yang D., Liu K., Zhang Y, & Huang L. (2022). Mallotucin D, a Clerodane Diterpenoid from Croton crassifolius, Suppresses HepG2 Cell Growth via Inducing Autophagic Cell Death and Pyroptosis. International Journal of Molecular Sciences, 23(22), 14217.
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7

Quantifying PDGF-induced Renal VSMC Proliferation

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Human renal VSMCs at passages 3–8 were seeded onto poly-l-lysine coated coverslips placed in 12 mm wells at a density of 20,000 cells/well. Cells were maintained in control medium for 24 hours and synchronized in serum free (SF) medium during 48 hours. Then, PDGF (20 ng/ml) was added for an additional 24 hours period. Tungstate and/or the BK channel blocker IbTX were added one hour before and remained present during PDGF stimulation. The percentage of cells at the S phase was quantified using EdU (5-ethynyl-2´-deoxyuridine) incorporation for another 6 hours with a commercial kit (Click-iT EdU Imaging Cell Proliferation Assay, Invitrogen). Finally, cells were incubated with Hoechst before mounting with Vectashield (Vector Laboratories Inc.,Burlingame, CA). EdU incorporation was visualized with an immunofluorescence microscopy (Nikon) at the corresponding wavelength depending on the Alexa Fluor used and was expressed as the percentage of the total cell number stained with Hoechst. In each experiment, this percentage was an average of 10 to 20 high power fields per coverslip. Triplicates were made for each condition.
Fernández-Mariño A.I., Cidad P., Zafra D., Nocito L., Domínguez J., Oliván-Viguera A., Köhler R., López-López J.R., Pérez-García M.T., Valverde M.Á., Guinovart J.J, & Fernández-Fernández J.M. (2015). Tungstate-Targeting of BKαβ1 Channels Tunes ERK Phosphorylation and Cell Proliferation in Human Vascular Smooth Muscle. PLoS ONE, 10(2), e0118148.
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8

Immunofluorescence Localization of Endothelial and Fibroblast Markers

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The expression and the localization of different target proteins were observed using immunofluorescence methods. HUVECs and CFs were fixed with 4% paraformaldehyde, permeabilized and then blocked with 2% BSA. Thereafter, cells were incubated with primary antibodies against CD31 (Abcam, ab28364) and α‐SMA (Abcam, ab5694) in HUVECs, and α‐SMA (Abcam, ab5694) in CFs overnight at 4°C. After incubation with two different fluorescent secondary antibodies for 30 minutes at 37°C and staining with DAPI, cell slides were sealed using antifade mounting medium. Finally, images were obtained with immunofluorescence microscopy (Nikon).
Tian J., Zhang M., Suo M., Liu D., Wang X., Liu M., Pan J., Jin T, & An F. (2021). Dapagliflozin alleviates cardiac fibrosis through suppressing EndMT and fibroblast activation via AMPKα/TGF‐β/Smad signalling in type 2 diabetic rats. Journal of Cellular and Molecular Medicine, 25(16), 7642-7659.
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9

Mitochondrial Membrane Potential Assay

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JC-1 dye (Thermo Fisher SCIENTIFIC, T3168) is a membrane permeable dye to determine mitochondrial membrane potential. It can selectively enter the mitochondria where it reversibly changes color with membrane potential changing. Upon membrane polarization, the formation of JC-1 aggregates cause shifts in emitted light from 530 to 590 nm when excited at 488 nm. The treated cells were labeled with 10 μg/mL JC-1 dye and fluorescence was determined using BD FACSCanto™ II Cell Analyzer (BD Biosciences-US) or Immunofluorescence Microscopy (Nikon, Japan).
Sun X., Shu Y., Ye G., Wu C., Xu M., Gao R., Huang D, & Zhang J. (2021). Histone deacetylase inhibitors inhibit cervical cancer growth through Parkin acetylation-mediated mitophagy. Acta Pharmaceutica Sinica. B, 12(2), 838-852.
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10

Immunohistochemical Protein Detection

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Tumor tissue samples were embedded in paraffin and antigen retrieval was performed. Following the blockade of endogenous peroxidase activity, the samples were incubated with the primary antibodies of interest and the appropriate secondary antibodies and reacted with DAB Detection Kit (Servicebio, G1212). The immunoreactive staining of proteins in tumor tissue was detected using Immunofluorescence Microscopy (Nikon, Japan).
Sun X., Shu Y., Ye G., Wu C., Xu M., Gao R., Huang D, & Zhang J. (2021). Histone deacetylase inhibitors inhibit cervical cancer growth through Parkin acetylation-mediated mitophagy. Acta Pharmaceutica Sinica. B, 12(2), 838-852.
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