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Immunofluorescence

Immunofluorescence is a powerful technique that utilizes fluorescent dyes to visualize and localize specific target proteins within cells or tissue samples.
This method involves the use of antibodies that are conjugated with fluorescent molecules, allowing researchers to detect and analyze the distribution and abundance of proteins of interest.
Immunofluorescence is widely employed in various fields of biomedical research, such as cell biology, immunology, and neuroscience, to study a wide range of biological processes and disease mechanisms.
It offers high specificity, sensitivity, and the ability to observe subcellular structures and interactions in a spatial context.
The power of immunofluorescence lies in its versatility, enabling researchers to gain valuable insights into the complex dynamics of cellular and molecular systems with precison.
However, optimizing immunofluorescence experiments can be challenging, requiring careful selection of antibodies, fluorophores, and imaging techniques to ensure reproducible and meaningful results.
The PubCompare.ai platform aims to simplify this process by providing AI-driven tools to help researchers identify the best protocols and reagents, thereby taking the guesswork out of their immunofluorescence research and unlocking the full potential of this essential technique.

Most cited protocols related to «Immunofluorescence»

1
Monocyte differentiation and activation
PBMC (1 × 106/mL) are cultured in RPMI 1640 supplemented with 10% human serum, 2 mM L-glutamine, and 1% penicillin (Invitrogen Ltd, Paisley, UK) and incubated at 37°C in a humidified 5% CO2 atmosphere for 2 h in a 12-well plate. After 2 h, non-adhering PBMCs are harvested and discarded; monocytes (adhering cells) are culture in medium alone (unstimulated) or primed with 2 μg/mL LPS for 2 h (Sigma–Aldrich, St. Louis, MO) before stimulation with Nigericine (5 μM) (Sigma–Aldrich) for 1 h at 37°C in a humidified 5% CO2 atmosphere. Adhering cells (monocytes) are then collected by trypsin treatment and prepared for FlowSight analysis by immunofluorescence staining as THP1-derived macrophage (see above).
Cossarizza A., Chang H.D., Radbruch A., Acs A., Adam D., Adam-Klages S., Agace W.W., Aghaeepour N., Akdis M., Allez M., Almeida L.N., Alvisi G., Anderson G., Andrä I., Annunziato F., Anselmo A., Bacher P., Baldari C.T., Bari S., Barnaba V., Barros-Martins J., Battistini L., Bauer W., Baumgart S., Baumgarth N., Baumjohann D., Baying B., Bebawy M., Becher B., Beisker W., Benes V., Beyaert R., Blanco A., Boardman D.A., Bogdan C., Borger J.G., Borsellino G., Boulais P.E., Bradford J.A., Brenner D., Brinkman R.R., Brooks A.E., Busch D.H., Büscher M., Bushnell T.P., Calzetti F., Cameron G., Cammarata I., Cao X., Cardell S.L., Casola S., Cassatella M.A., Cavani A., Celada A., Chatenoud L., Chattopadhyay P.K., Chow S., Christakou E., Čičin-Šain L., Clerici M., Colombo F.S., Cook L., Cooke A., Cooper A.M., Corbett A.J., Cosma A., Cosmi L., Coulie P.G., Cumano A., Cvetkovic L., Dang V.D., Dang-Heine C., Davey M.S., Davies D., De Biasi S., Del Zotto G., Cruz G.V., Delacher M., Bella S.D., Dellabona P., Deniz G., Dessing M., Di Santo J.P., Diefenbach A., Dieli F., Dolf A., Dörner T., Dress R.J., Dudziak D., Dustin M., Dutertre C.A., Ebner F., Eckle S.B., Edinger M., Eede P., Ehrhardt G.R., Eich M., Engel P., Engelhardt B., Erdei A., Esser C., Everts B., Evrard M., Falk C.S., Fehniger T.A., Felipo-Benavent M., Ferry H., Feuerer M., Filby A., Filkor K., Fillatreau S., Follo M., Förster I., Foster J., Foulds G.A., Frehse B., Frenette P.S., Frischbutter S., Fritzsche W., Galbraith D.W., Gangaev A., Garbi N., Gaudilliere B., Gazzinelli R.T., Geginat J., Gerner W., Gherardin N.A., Ghoreschi K., Gibellini L., Ginhoux F., Goda K., Godfrey D.I., Goettlinger C., González-Navajas J.M., Goodyear C.S., Gori A., Grogan J.L., Grummitt D., Grützkau A., Haftmann C., Hahn J., Hammad H., Hämmerling G., Hansmann L., Hansson G., Harpur C.M., Hartmann S., Hauser A., Hauser A.E., Haviland D.L., Hedley D., Hernández D.C., Herrera G., Herrmann M., Hess C., Höfer T., Hoffmann P., Hogquist K., Holland T., Höllt T., Holmdahl R., Hombrink P., Houston J.P., Hoyer B.F., Huang B., Huang F.P., Huber J.E., Huehn J., Hundemer M., Hunter C.A., Hwang W.Y., Iannone A., Ingelfinger F., Ivison S.M., Jäck H.M., Jani P.K., Jávega B., Jonjic S., Kaiser T., Kalina T., Kamradt T., Kaufmann S.H., Keller B., Ketelaars S.L., Khalilnezhad A., Khan S., Kisielow J., Klenerman P., Knopf J., Koay H.F., Kobow K., Kolls J.K., Kong W.T., Kopf M., Korn T., Kriegsmann K., Kristyanto H., Kroneis T., Krueger A., Kühne J., Kukat C., Kunkel D., Kunze-Schumacher H., Kurosaki T., Kurts C., Kvistborg P., Kwok I., Landry J., Lantz O., Lanuti P., LaRosa F., Lehuen A., LeibundGut-Landmann S., Leipold M.D., Leung L.Y., Levings M.K., Lino A.C., Liotta F., Litwin V., Liu Y., Ljunggren H.G., Lohoff M., Lombardi G., Lopez L., López-Botet M., Lovett-Racke A.E., Lubberts E., Luche H., Ludewig B., Lugli E., Lunemann S., Maecker H.T., Maggi L., Maguire O., Mair F., Mair K.H., Mantovani A., Manz R.A., Marshall A.J., Martínez-Romero A., Martrus G., Marventano I., Maslinski W., Matarese G., Mattioli A.V., Maueröder C., Mazzoni A., McCluskey J., McGrath M., McGuire H.M., McInnes I.B., Mei H.E., Melchers F., Melzer S., Mielenz D., Miller S.D., Mills K.H., Minderman H., Mjösberg J., Moore J., Moran B., Moretta L., Mosmann T.R., Müller S., Multhoff G., Muñoz L.E., Münz C., Nakayama T., Nasi M., Neumann K., Ng L.G., Niedobitek A., Nourshargh S., Núñez G., O’Connor J.E., Ochel A., Oja A., Ordonez D., Orfao A., Orlowski-Oliver E., Ouyang W., Oxenius A., Palankar R., Panse I., Pattanapanyasat K., Paulsen M., Pavlinic D., Penter L., Peterson P., Peth C., Petriz J., Piancone F., Pickl W.F., Piconese S., Pinti M., Pockley A.G., Podolska M.J., Poon Z., Pracht K., Prinz I., Pucillo C.E., Quataert S.A., Quatrini L., Quinn K.M., Radbruch H., Radstake T.R., Rahmig S., Rahn H.P., Rajwa B., Ravichandran G., Raz Y., Rebhahn J.A., Recktenwald D., Reimer D., e Sousa C.R., Remmerswaal E.B., Richter L., Rico L.G., Riddell A., Rieger A.M., Robinson J.P., Romagnani C., Rubartelli A., Ruland J., Saalmüller A., Saeys Y., Saito T., Sakaguchi S., de-Oyanguren F.S., Samstag Y., Sanderson S., Sandrock I., Santoni A., Sanz R.B., Saresella M., Sautes-Fridman C., Sawitzki B., Schadt L., Scheffold A., Scherer H.U., Schiemann M., Schildberg F.A., Schimisky E., Schlitzer A., Schlosser J., Schmid S., Schmitt S., Schober K., Schraivogel D., Schuh W., Schüler T., Schulte R., Schulz A.R., Schulz S.R., Scottá C., Scott-Algara D., Sester D.P., Shankey T.V., Silva-Santos B., Simon A.K., Sitnik K.M., Sozzani S., Speiser D.E., Spidlen J., Stahlberg A., Stall A.M., Stanley N., Stark R., Stehle C., Steinmetz T., Stockinger H., Takahama Y., Takeda K., Tan L., Tárnok A., Tiegs G., Toldi G., Tornack J., Traggiai E., Trebak M., Tree T.I., Trotter J., Trowsdale J., Tsoumakidou M., Ulrich H., Urbanczyk S., van de Veen W., van den Broek M., van der Pol E., Van Gassen S., Van Isterdael G., van Lier R.A., Veldhoen M., Vento-Asturias S., Vieira P., Voehringer D., Volk H.D., von Borstel A., von Volkmann K., Waisman A., Walker R.V., Wallace P.K., Wang S.A., Wang X.M., Ward M.D., Ward-Hartstonge K.A., Warnatz K., Warnes G., Warth S., Waskow C., Watson J.V., Watzl C., Wegener L., Weisenburger T., Wiedemann A., Wienands J., Wilharm A., Wilkinson R.J., Willimsky G., Wing J.B., Winkelmann R., Winkler T.H., Wirz O.F., Wong A., Wurst P., Yang J.H., Yang J., Yazdanbakhsh M., Yu L., Yue A., Zhang H., Zhao Y., Ziegler S.M., Zielinski C., Zimmermann J, & Zychlinsky A. (2019). Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). European journal of immunology, 49(10), 1457-1973.
Publication 2019
Atmosphere Cells Culture Media Glutamine Homo sapiens Immunofluorescence Macrophage Monocytes Penicillins Serum Trypsin
2
Whole Genome Sequencing of Tumor and Blood Samples
Human tumour and matched blood samples were obtained with informed consent through an institutional review board approved protocol at St Jude Children’s Research Hospital. Whole genome sequencing (WGS) and analysis of WGS data were performed as previously described50 (link). Details of sequence coverage, custom capture and other validation procedures are provided in Supplementary Information (Supplementary Tables 12-15). Sequence and SNP array data were deposited in dbGaP (dbGaP accession number: phs000409, SRA accession number: SRP008292). Immunohistochemistry and immunofluorescence of human and mouse tissues were performed using routine techniques and primary antibodies of the appropriate tissues as described (Supplementary Methods). Medulloblastoma mRNA and DNA profiles were generated using Affymetrix U133v2 and SNP 6.0 arrays, respectively (Supplementary Methods). Reverse transcriptase Real Time-PCR analysis of genes targeted in mouse LRLPs by shRNAs were performed as described previously32 (link). LRLPs were isolated and transduced with indicated lentiviruses in stem cell cultures or targeted in utero with shRNAs or mutant cDNA sequences by electroporation as described (Supplementary Information)5 (link). Mice harbouring a cre-inducible Pik3caE545K allele were generated using homologous recombination: A lox-puro-STOP-lox cassette was introduced immediately upstream of the exon containing the initiation codon, exon 9 was replaced with an exon containing the E545K mutation. Pik3caE545K mice were bred with Blbp-Cre, Ctnnb1lox(ex3)/lox(ex3) and Tp53flx/flx mice to generate progeny of the appropriate genotype and subjected to clinical surveillance.
Robinson G., Parker M., Kranenburg T.A., Lu C., Chen X., Ding L., Phoenix T.N., Hedlund E., Wei L., Zhu X., Chalhoub N., Baker S.J., Huether R., Kriwacki R., Curley N., Thiruvenkatam R., Wang J., Wu G., Rusch M., Hong X., Beckford J., Gupta P., Ma J., Easton J., Vadodaria B., Onar-Thomas A., Lin T., Li S., Pounds S., Paugh S., Zhao D., Kawauchi D., Roussel M.F., Finkelstein D., Ellison D.W., Lau C.C., Bouffet E., Hassall T., Gururangan S., Cohn R., Fulton R.S., Fulton L.L., Dooling D.J., Ochoa K., Gajjar A., Mardis E.R., Wilson R.K., Downing J.R., Zhang J, & Gilbertson R.J. (2012). Novel mutations target distinct subgroups of medulloblastoma. Nature, 488(7409), 43-48.
Publication 2012
Alleles Antibodies BLOOD Cell Culture Techniques Child Codon, Initiator DNA, Complementary Electroporation Ethics Committees, Research Exons Genes Genotype Homologous Recombination Homo sapiens Immunofluorescence Immunohistochemistry Lentivirus Medulloblastoma Mus Mutation Neoplasms Real-Time Polymerase Chain Reaction RNA, Messenger RNA-Directed DNA Polymerase Short Hairpin RNA Stem, Plant Stem Cells Tissues Uterus
3
Autophagy Regulation of Lipid Metabolism
The hepatocyte cell line RALA255-10G was cultured under nontransformed conditions, as described previously20 (link). Wild-type and Atg5−/− mouse embryonic fibroblasts provided by N. Mizushima21 (link) were cultured as described previously22 (link). TG content was determined by the Trig/GB Kit (Roche Diagnostics), cholesterol content by the Amplex Red Cholesterol Assay (Invitrogen), fatty acid β-oxidation by a modification of a previously used method23 (link), and TG decay in cells radiolabelled with [14C]oleate and TG synthesis by standard methods12 (link). shRNAs were cloned into pSUPER (Ambion) and then pCCL.sin.PPT.hPGK.GFPWpre24 (link). Protein isolation and western blotting were performed as described previously25 (link). Fluorescence microscopy for BODIPY 493/503 (Invitrogen) and immunofluorescence were performed as described previously26 (link). Atg7F/F mice4 (link) were crossed with Alb-Cre mice27 (link) to generate Atg7F/F-Alb-Cre mice. Some animals were fed a high-fat diet (60% kcal in fat; Research Diets, D12492). Electron microscopy and immunogold labelling were performed as described previously26 (link). LDs from mouse livers were isolated by sucrose density gradient centrifugation28 and autophagic vacuoles and lysosomes by centrifugation in metrizamide discontinuous density gradients29 (link).
Singh R., Kaushik S., Wang Y., Xiang Y., Novak I., Komatsu M., Tanaka K., Cuervo A.M, & Czaja M.J. (2009). Autophagy regulates lipid metabolism. Nature, 458(7242), 1131-1135.
Publication 2009
4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene Anabolism Animals Autophagosome Biological Assay Cell Lines Cells Centrifugation Cholesterol Diagnosis Diet Diet, High-Fat Electron Microscopy Embryo Fatty Acids Fibroblasts formaldehyde-serum albumin Hepatocyte Immunofluorescence isolation Liver Lysosomes Metrizamide Mice, Laboratory Microscopy, Fluorescence Oleate Proteins Short Hairpin RNA Sucrose
4
Zebrafish Functional Genomics Protocols

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Publication 2011
Animals, Transgenic Biological Assay Cell Culture Techniques Cells Flow Cytometry Immunofluorescence Immunoprecipitation, Chromatin In Situ Hybridization Luciferases Microscopy, Confocal Morpholinos Reverse Transcriptase Polymerase Chain Reaction Tissues Transfection Western Blot Zebrafish
5
Detecting Apoptosis and Oxidative Stress in Yeast
Electron microscopy, annexin V labeling, and DAPI staining were performed as described previously (Madeo et al., 1997 (link)). For the TdT-mediated dUTP nick end labeling (TUNEL) test, cells were prepared as described (Madeo et al., 1997 (link)), and the DNA ends were labeled using the In Situ Cell Death Detection Kit, POD (Boehringer Mannheim). Yeast cells were fixed with 3.7% formaldehyde, digested with lyticase, and applied to a polylysine-coated slide as described for immunofluorescence (Adams and Pringle, 1984 (link)). The slides were rinsed with PBS and incubated with 0.3% H2O2 in methanol for 30 min at room temperature to block endogenous peroxidases. The slides were rinsed with PBS, incubated in permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) for 2 min on ice, rinsed twice with PBS, incubated with 10 μl TUNEL reaction mixture (terminal deoxynucleotidyl transferase 200 U/ml, FITC-labeled dUTP 10 mM, 25 mM Tris-HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride; Boehringer Mannheim) for 60 min at 37°C, and then rinsed 3× with PBS. For the detection of peroxidase, cells were incubated with 10 μl Converter-POD (anti-FITC antibody, Fab fragment from sheep, conjugated with horseradish peroxidase) for 30 min at 37°C, rinsed 3× with PBS, and then stained with DAB-substrate solution (Boehringer Mannheim) for 10 min at room temperature. A coverslip was mounted with a drop of Kaiser's glycerol gelatin (Merck). As staining intensity varies, only samples from the same slide were compared.
Free intracellular radicals were detected with dihydrorhodamine 123, dichlorodihydrofluorescein diacetate (dichlorofluorescin diacetate), or dihydroethidium (hydroethidine; Sigma Chemical Co.). Dihydrorhodamine 123 was added ad-5 μg per ml of cell culture from a 2.5-mg/ml stock solution in ethanol and cells were viewed without further processing through a rhodamine optical filter after a 2-h incubation. Dichlorodihydrofluorescein diacetate was added ad-10 μg per ml of cell culture from a 2.5 mg/ml stock solution in ethanol and cells were viewed through a fluorescein optical filter after a 2-h incubation. Dihydroethidium was added ad-5 μg per ml of cell culture from a 5 mg/ml aqueous stock solution and cells were viewed through a rhodamine optical filter after a 10-min incubation. For flow cytometric analysis, cells were incubated with dihydrorhodamine 123 for 2 h and analyzed using a FACS® Calibur (Becton Dickinson) at low flow rate with excitation and emission settings of 488 and 525–550 nm (filter FL1), respectively.
Free spin trap reagents N-tert-butyl-α−phenylnitrone (PBN; Sigma-Aldrich) and 3,3,5,5,-tetramethyl-pyrroline N-oxide (TMPO; Sigma-Aldrich) were added directly to the cell cultures as 10-mg/ml aqueous stock solutions. Viability was determined as the portion of cell growing to visible colonies within 3 d.
To determine frequencies of morphological phenotypes (TUNEL, Annexin V, DAPI, dihydrorhodamine 123), at least 300 cells of three independent experiments were evaluated.
Madeo F., Fröhlich E., Ligr M., Grey M., Sigrist S.J., Wolf D.H, & Fröhlich K.U. (1999). Oxygen Stress: A Regulator of Apoptosis in Yeast. The Journal of Cell Biology, 145(4), 757-767.
Publication 1999
3,3,5,5-tetramethyl-1-pyrroline N-oxide Annexin A5 Antibodies, Anti-Idiotypic Cacodylate Cardiac Arrest Cell Culture Techniques Cell Death Cells cobaltous chloride DAPI deoxyuridine triphosphate dichlorofluorescin dihydroethidium dihydrorhodamine 123 DNA Nucleotidylexotransferase Domestic Sheep Electron Microscopy Ethanol Flow Cytometry Fluorescein Fluorescein-5-isothiocyanate Formaldehyde Free Radicals Gelatins Glycerin Horseradish Peroxidase hydroethidine Immunofluorescence Immunoglobulins, Fab In Situ Nick-End Labeling lyticase Methanol Oxides Peroxidase Peroxidases Peroxide, Hydrogen Phenotype Polylysine Protoplasm pyrroline Rhodamine Sodium Sodium Citrate TERT protein, human Triton X-100 Tromethamine Yeast, Dried

Most recents protocols related to «Immunofluorescence»

1
Dapansutrile Reduces Metastatic Spread
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Example 3

The mechanisms of metastasis involve a complex multi-step process of detachment from the primary tumor site, intravasation into circulation, survival in the circulation, extravasation from circulation, attachment at a secondary site, and development of secondary tumor sites.

To determine whether dapansutrile reduces tissue invasion and metastasis, B16F10-GFP labeled cells were injected intravenously in mice fed with standard or OLT1177® diets.

Immunofluorescence analysis of the lungs and the livers showed reduced number of GFP+ cells in mice that received OLT1177® compared to the standard diet. In FIG. 3A, the number of GFP+ cells in the lungs is reduced by 66% (p<0.0001) by dapansutrile treatment. A similar reduction (−60%; p<0.001) in the number of GFP+ B1610 cells was observed in the liver (FIG. 3B). Together, the reductions in the number of metastatic cells in the lung and liver indicate that dapansutrile reduces tissue invasion and reduces the metastasis in both liver and in lung.

US11857529B2. Methods for treating melanoma (2024-01-02). Olatec Therageutics, Inc. [US]. Inventors: Carlo Marchetti [US], Charles A. Dinarello [US].
Patent 2024
dapansutrile Diet Immunofluorescence Liver Lung Mus Neoplasm Metastasis Neoplasms Neoplasms by Site OLT1177 Tissues
2
Colocalization of Antibodies in B-cells
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Example 4

To determine where 2F2-grafted “humanized” antibodies and antibody variants are delivered upon internalization into the cell, colocalization studies of the anti-CD79b antibodies internalized into B-cell lines may be assessed in Ramos cell lines. LAMP-1 is a marker for late endosomes and lysosomes (Kleijmeer et al., Journal of Cell Biology, 139(3): 639-649 (1997); Hunziker et al., Bioessays, 18:379-389 (1996); Mellman et al., Annu. Rev. Dev. Biology, 12:575-625 (1996)), including MHC class II compartments (MIICs), which is a late endosome/lysosome-like compartment. HLA-DM is a marker for MIICs.

Ramos cells are incubated for 3 hours at 37° C. with 1 μg/ml 2F2-grafted “humanized” antibodies and antibody variants, FcR block (Miltenyi) and 25 μg/ml Alexa647-Transferrin (Molecular Probes) in complete carbonate-free medium (Gibco) with the presence of 10 μg/ml leupeptin (Roche) and 5 μM pepstatin (Roche) to inhibit lysosomal degradation. Cells are then washed twice, fixed with 3% paraformaldehyde (Electron Microscopy Sciences) for 20 minutes at room temperature, quenched with 50 mM NH4Cl (Sigma), permeabilized with 0.4% Saponin/2% FBS/1% BSA for 20 minutes and then incubated with 1 μg/ml Cy3 anti-mouse (Jackson Immunoresearch) for 20 minutes. The reaction is then blocked for 20 minutes with mouse IgG (Molecular Probes), followed by a 30 minute incubation with Image-iT FX Signal Enhancer (Molecular Probes). Cells are finally incubated with Zenon Alexa488-labeled mouse anti-LAMP1 (BD Pharmingen), a marker for both lysosomes and MIIC (a lysosome-like compartment that is part of the MHC class II pathway), for 20 minutes, and post-fixed with 3% PFA. Cells are resuspended in 20 μl saponin buffer and allowed to adhere to poly-lysine (Sigma) coated slides prior to mounting a coverglass with DAPI-containing VectaShield (Vector Laboratories). For immunofluorescence of the MIIC or lysosomes, cells are fixed, permeabilized and enhanced as above, then co-stained with Zenon labeled Alexa555-HLA-DM (BD Pharmingen) and Alexa488-Lamp1 in the presence of excess mouse IgG as per the manufacturer's instructions (Molecular Probes).

Accordingly, colocalization of 2F2-grafted “humanized” antibodies or antibody variants with MIIC or lysosomes of B-cell lines as assessed by immunofluorescence may indicate the molecules as excellent agents for therapy of tumors in mammals, including B-cell associated cancers, such as lymphomas (i.e. Non-Hodgkin's Lymphoma), leukemias (i.e. chronic lymphocytic leukemia), and other cancers of hematopoietic cells.

US11866496B2. Humanized anti-CD79B antibodies and immunoconjugates and methods of use (2024-01-09). Genentech, Inc. [US]. Inventors: Yvonne Chen [US], Mark Dennis [US], Kristi Elkins [US], Jagath Reddy Junutula [US], Andrew Polson [US], Bing Zheng [US].
Patent 2024
Alexa Fluor 647 Anti-Antibodies Antibodies, Monoclonal, Humanized B-Lymphocytes Buffers Carbonates CD79B protein, human Cell Lines Cells Chronic Lymphocytic Leukemia Cloning Vectors DAPI Electron Microscopy Endosomes Genes, MHC Class II Hematopoietic Neoplasms Immunofluorescence Immunoglobulins Leukemia leupeptin Lymphoma Lymphoma, Non-Hodgkin Lysine lysosomal-associated membrane protein 1, human Lysosomes Malignant Neoplasms Mammals Molecular Probes Mus Neoplasms paraform pepstatin Poly A Saponin Therapeutics Transferrin
3
Mitochondria, Cytoskeleton Immunofluorescence
Procedures of HCFs (5–10 passages) prepared for immunofluorescent staining of mitochondria, β-actin, and actin-related protein-2 (Arp2) are detailed in Additional file 2.
Hsu C.C., Wang J.S., Shyu Y.C., Fu T.C., Juan Y.H., Yuan S.S., Wang C.H., Yeh C.H., Liao P.C., Wu H.Y, & Hsu P.H. (2023). Hypermethylation of ACADVL is involved in the high-intensity interval training-associated reduction of cardiac fibrosis in heart failure patients. Journal of Translational Medicine, 21, 187.
Publication 2023
Actin-Related Protein 2 Actins Immunofluorescence Mitochondria
4
Quantifying Total Cellular Protein via TIRFM
For detection of total cellular protein (all immunofluorescence experiments), after treatments, as indicated, cells were fixed with 4% paraformaldehyde for 30 min, followed by quenching of fixative in 100 mM glycine, cell permeabilization in 0.1% Triton X-100 (all solutions made in PBS), and then blocking in 3% BSA (Thermo Fisher Scientific). Subsequently, cells were stained with primary and secondary antibodies as indicated and retained within an aqueous medium for imaging by TIRFM.
Rahmani S., Ahmed H., Ibazebo O., Fussner-Dupas E., Wakarchuk W.W, & Antonescu C.N. (2023). O-GlcNAc transferase modulates the cellular endocytosis machinery by controlling the formation of clathrin-coated pits. The Journal of Biological Chemistry, 299(3), 102963.
Publication 2023
Aftercare Antibodies Cells Fixatives Glycine Immunofluorescence paraform Proteins Triton X-100
5
Wear Particle-Induced Calvarial Osteolysis Model

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Publication 2023
Calvaria Cranium Diet Immunofluorescence Immunohistochemistry Males Mice, Inbred C57BL Mus Operative Surgical Procedures Osteolysis paraform Pentobarbital Sodium Periosteum

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Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
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DAPI is a fluorescent dye that binds strongly to adenine-thymine (A-T) rich regions in DNA. It is commonly used as a nuclear counterstain in fluorescence microscopy to visualize and locate cell nuclei.
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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium formulated to support the growth and maintenance of a variety of cell types, including mammalian cells. It provides essential nutrients, amino acids, vitamins, and other components necessary for cell proliferation and survival in an in vitro environment.
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Alexa Fluor 488 is a fluorescent dye used in various biotechnological applications. It has an excitation maximum at 495 nm and an emission maximum at 519 nm, producing a green fluorescent signal. Alexa Fluor 488 is known for its brightness, photostability, and pH-insensitivity, making it a popular choice for labeling biomolecules in biological research.
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Penicillin streptomycin by Thermo Fisher Scientific
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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.
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Triton x 100 by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, China, Japan, France, Canada, Sao Tome and Principe, Switzerland, Macao, Poland, Spain, Australia, India, Belgium, Israel, Sweden, Ireland, Denmark, Brazil, Portugal, Panama, Netherlands, Hungary, Czechia, Austria, Norway, Slovakia, Singapore, Argentina, Mexico, Senegal
Triton X-100 is a non-ionic surfactant commonly used in various laboratory applications. It functions as a detergent and solubilizing agent, facilitating the solubilization and extraction of proteins and other biomolecules from biological samples.
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Lipofectamine 3000 by Thermo Fisher Scientific
Sourced in United States, China, Germany, Japan, United Kingdom, France, Canada, Italy, Australia, Switzerland, Denmark, Spain, Singapore, Belgium, Lithuania, Israel, Sweden, Austria, Moldova, Republic of, Greece, Azerbaijan, Finland
Lipofectamine 3000 is a transfection reagent used for the efficient delivery of nucleic acids, such as plasmid DNA, siRNA, and mRNA, into a variety of mammalian cell types. It facilitates the entry of these molecules into the cells, enabling their expression or silencing.
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Lipofectamine rnaimax by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom, Germany, Japan, France, Canada, Switzerland, Denmark, Belgium, Italy, Australia, Singapore, Spain, Colombia, Sweden, Netherlands, New Zealand, Poland, Pakistan, Lithuania
Lipofectamine RNAiMAX is a transfection reagent designed for efficient delivery of small interfering RNA (siRNA) and short hairpin RNA (shRNA) into a wide range of cell types. It is a cationic lipid-based formulation that facilitates the uptake of these nucleic acids into the target cells.

More about "Immunofluorescence"

Immunofluorescence (IF) is a powerful imaging technique that utilizes fluorescent dyes and antibodies to visualize and localize specific target proteins within cells or tissue samples.
This method, also known as fluorescent immunoassay or immunocytochemistry, involves the use of fluorophore-conjugated antibodies that bind to the proteins of interest, enabling researchers to detect and analyze their distribution and abundance.
Immunofluorescence finds widespread applications in various fields of biomedical research, such as cell biology, immunology, neuroscience, and developmental biology, to study a wide range of biological processes and disease mechanisms.
It offers high specificity, sensitivity, and the ability to observe subcellular structures and interactions in a spatial context, making it an essential tool for understanding complex cellular and molecular systems.
The success of immunofluorescence experiments relies on the careful selection and optimization of various components, including antibodies, fluorescent labels (e.g., Alexa Fluor 488), and imaging techniques.
Reagents like Lipofectamine 2000, Lipofectamine 3000, and Lipofectamine RNAiMAX can be used for cell transfection and gene silencing, while DAPI, a fluorescent DNA stain, is often used to visualize nuclei.
Culture media like DMEM and supplements such as FBS and Penicillin/streptomycin are also commonly used in immunofluorescence experiments.
Proper sample preparation, including fixation, permeabilization with Triton X-100, and blocking, is crucial for ensuring specific and reproducible results.
The PubCompare.ai platform can help researchers navigate this process by providing AI-driven tools to identify the best protocols and reagents, taking the guesswork out of their immunofluorescence research and unlocking the full potential of this essential technique.