Lc3 primary antibody in 1% normal serum or bsa in 1x pbs 5. During autophagy, lc3i is converted to lc3ii through lipidation by a ubiquitinlike system involving atg7 and atg3 that allows for lc3 to become associated with autophagic vesicles 610. The presence of lc3 in autophagosomes and the conversion of lc3 to the lower migrating form, lc3ii, have been used as indicators of autophagy 11. Immunofluorescence an overview sciencedirect topics. It is also suitable for analysis of cells expressing gfplc3. Antimicrotubuleassociated protein 1 light chain 3 alpha, antimicrotubuleassociated protein 1 light chain 3 beta, antimicrotubuleassociated proteins 1a1b light chain. Provided below are standard protocols that you may find useful for product applications. Immunofluorescence protocols thermo fisher scientific us. View protocol download pdf antibodies, kits and reagents for crisp and bright stainings in an immunofluorescence if experiment a primary antibody binds specifically to a protein of interest present in a sample e. Recombinant antilc3b antibody epr18709 autophagosome. Mitophagy analysis monitoring mitochondrial protein degradation in parkinmediated mitophagy saori r. It is also suitable for analysis of cells expressing gfp lc3.
Immunofluorescence is a powerful tool for elucidating the complex signaling events that underlie biological processes and disease. A quantitative assay for the monitoring of autophagosome. The following protocol describes three simple and commonly used protocols for measuring autophagy in cells. In situ immunofluorescent staining of autophagy in muscle. Lc3 antibody autophagosome marker superior for western. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of lc3, an autophagy marker, and myod, a myogenic lineage marker, in muscle tissue sections from control and injured mice.
Categories immunology tags direct immunofluorescence protocol, direct. All lc3 isoforms undergo posttranslational modifications, especially pe conjugation lipidation during autophagy. Autophagy is a common morphological feature in dying cells, and therefore it was presumed for. Use separate tubs for antibodies and negative control slides.
Blocking solution 1% bsa or 10% fcs fetal calf serum in. Then paraformaldehyde is quenched by washing coverslips twice with 100 mm in pbs. Also monoclonal and polyclonal antibodies do not necessarily react with the same antigenic form of a component. Herein, this protocol describes the predominant method used for the research of autophagy, which mainly focuses on the immunofluorescence staining of cellular lc3, p62, and ulk1 in response to normoxia and hypoxia, by presenting the detailed materials required and methodology.
Hela and neuro2a cells were treated with or without. Immunofluorescence protocol united states biological. For more information on western blotting, see our western blot. Thus, lysosomal turnover of the autophagosomal marker lc3 ii reflects starvationinduced autophagic activity, and detecting lc3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy and autophagyrelated processes, including autophagic cell death.
Protocol for immunofluorescence staining of adhesion cells. Lc3 antibody 1712d nbp246888 western blot image of monoclonal antilc3iii clone 1712d. Lc3 antibody apg8b nterm additional information gene id 81631 other names microtubuleassociated proteins 1a1b light chain 3b, autophagyrelated protein lc3 b, autophagyrelated ubiquitinlike modifier lc3 b, map1 light chain 3like protein 2, map1amap1b light chain 3 b, map1amap1b lc3 b, microtubuleassociated protein 1 light. Immunofluorescence is an assay which is used primarily on biological samples and is classically defined as a procedure to detect antigens in cellular contexts using antibodies. Such issues include the effects of the heterogeneity of transfection efficiency and gfplc3 expression levels between cells, the induction of autophagy by both the transfection protocol and overexpression of gfplc3 and finally by the propensity of gfplc3 to associate with protein aggregates already present in cells as well as to self. Immunofluorescence and immunocytochemistry protocol. Western blot blocking peptides dot blot immunohistochemistry immunofluorescence immunoprecipitation flow cytomety cell culture line. Pdf immunofluorescence staining protocols for major autophagy.
Optimization of concentration or incubation condition of the primary antibody and the secondary antibody for your own specimen is necessary. Although staining for lc3 by immunohistochemistry has been reported, 2731 to the best of our knowledge no protocol has so far been published which details a method for immunohistochemistry of lc3 on human and mouse tissue sections. Protocol for immunofluorescence staining of adhesion cells this is provided as a general protocol. Thus, lysosomal turnover of the autophagosomal marker lc3ii reflects starvationinduced autophagic activity, and detecting lc3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy and autophagyrelated processes, including autophagic cell death. Monolayer cells are cultured and prepared on a coverslips before the immunofluorescence procedure. Since lc3 is the only protein identified on the inner and outer membranes of autophagosomes, map1lc3ab antibodies provide a quick detection method. Immunofluorescence or fluorescent antibody staining is an antigendetection test that is used primarily on frozen tissue sections, cell smears, or cultured cells. Please refer to the western blot protocol found on the product web.
Autophagy marker light chain 3 lc3 was originally identified as a subunit of. Noted the lc3a accumulation in the perinuclear area, while lc3b is distributed throughout the cytoplasm a17. This assay could be used for monitoring autophagosomes by flow cytometry utilizing immunostaining with the antibody against the lc3 protein. At the same time, lc3 ii in autolysosomal lumen is degraded. Inhibition of autophagy and lc3b antibody nb1002220. Knockout tested rabbit recombinant monoclonal lc3b antibody epr18709 autophagosome marker. Autophagy, lc3, acridine orange, immunofluorescence, western blot.
Immunofluorescence protocol university of pittsburgh. Autophagosome proteins lc3a, lc3b and lc3c have distinct. Mitophagy analysis monitoring mitochondrial protein. To support this commitment, each invitrogen antibody that is indicated for immunofluorescence applications has been tested using a protocol similar to that provided below. Microtubuleassociated protein light chain 3 lc3 is now widely used to monitor autophagy. Herein, this protocol describes the predominant method used for the research of autophagy, which mainly focuses on the immunofluorescence staining of cellular lc3, p62, and ulk1 in response to. Increase the manual exposure time of your camera or use the autoexposure option that will adjust the. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample.
It is expressed as the splice variants lc3a, lc3b, and lc3c which display unique tissue distribution. Pdf immunofluorescence staining protocols for major. During aut ophagy, lc3i is c onv ert ed t o lc3i i t hrough lipidat ion by a ubiquit inlik e s y s t em inv olv ing a t g7 and a t g3 t hat allows f or lc3 t o bec ome as s oc iat ed wit h aut ophagic v es ic les 610. As the protocol is the same and that i was not watching it for all the cells marked and positive, it seems to be random but i don. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the musc compartment, which. Immunofluorescence staining protocols for major autophagy. Validated in wb, ip, iccif and tested in mouse, rat, human.
Seed cells on sterile coverslips in 24 well plates in dmem supplemented with 10% volvol heatinactivated fbs and 50 u ml1 penicillinstreptomycin and 2 mm lglutamine, and. Immunofluorescence immunofluorescence is an antigenantibody reaction where the antibodies are tagged labelled with a fluorescent dye and the antigenantibody complex is visualized using ultraviolet fluorescent microscope. Immunofluorescence protocol paraformaldehyde fixed tissue, see below for nonfixed tissue notes before you begin. Lc3positive structures are prominent in autophagydeficient cells. Immunofluorescence staining protocols for major autophagy proteins including lc3, p62, and ulk1 in mammalian cells in response to. Immunofluorescence protocol adherent and suspension for.
Synthetic peptide corresponding to human lc3b n terminal. Assessing autophagic flux by measuring lc3, p62, and lamp1 colocalization using multispectral imaging flow cytometry. A confocal double immunofluorescence for lc3a green and lc3b red in various cell lines. In our hands, for best results, samples should be paraformaldehyde fixed and frozen using 2methylbutane cooled with liquid nitrogen see tissue fixation and freezing protocol. Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate fitc. This guide highlights critical steps in the immunofluorescence protocol and demonstrates how protocol changes can. Lc3i form of the protein from cells, fol lowed by flow cytometric detection of the autophagosomal membraneassociated fraction of lc3ii.
Synthetic peptide made to the n terminal region of human lc3, isoform b protein. Fluorochromes are dyes that absorb ultraviolet rays and emit visible light. Immunofluorescence microscopy of mitochondrial matrix proteins. A collection of immunofluorescence protocols for research, provided by life technologies. Antigen is detected through the binding to the sample matrix of specially modified, agentspecific antibodies. Immunocytochemistry and immunofluorescence protocol procedure for staining of cell cultures using immunofluorescence. The cells are washed with pbs twice and fixed with 3% paraformaldehyde at room temperature for 12 min.
Seed cells on sterile coverslips in 24 well plates in dmem supplemented with 10% volvol heatinactivated fbs and 50 u ml1 penicillinstreptomycin and 2 mm lglutamine, and maintain the cells at 37c with 5% co 2. Coat coverslips with polyethylineimine or polyllysine for 1 h at room temperature. There is no one perfect protocol of how to fix, stain, and analyze cells. Immunofluorescence is a valuable auxiliary diagnostic tool for autoimmune bullous diseases and inflammatory disorders, since their clinical and. Because of the low structural resolution of light microscopy it will be more important to preserve the antigenicity of a component. Lc3 i form of the protein from cells, fol lowed by flow cytometric detection of the autophagosomal membraneassociated fraction of lc3 ii. Examples of staining using traditional icc, icc if, and ihc with if based detection. Since there is an increasing demand to analyze autophagy in human biopsies and in transgenic animal models, we. Fixative 4% formaldehyde in pbs freshly prepared 3. Assessing autophagic flux by measuring lc3, p62, and lamp1. Lc3 immunostaining taki nishimura, noboru mizushima. Inhibition of autophagy and lc3b antibody nb1002220 immunocytochemistry icc materials chloroquine diphosphate cq 10 mm in dh2o 1x pbs 4% paraformaldehyde in 1x pbs make fresh 1x pbs with 0. Immunocytochemistry icc refers to immunostaining of cultured cell lines or primary cells including smears. Ihc wholemount, flow cyt, ihcfr, wb, iccif more details.
Here we describe basic protocols to assay for endogenous lc3ii by immunoblotting, immunoprecipitation, and immunofluorescence. There are two major types of immunofluorescence staining methods. Immunocytochemistry and immunofluorescence protocol. Immunofluorescence protocol adherent and suspension for application testing. This observation questions the reliability of lc3immunofluorescence assays in cells with compromised autophagy. Immunocytochemistry icc handbook novus biologicals. One approach is to detect lc3 conversion lc3i to lc3ii by immunoblot analysis because the amount of lc3ii is clearly correlated with the number of autophagosomes.
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