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LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #13173
Confocal immunofluorescent analysis of HeLa cells, chloroquine-treated (50 μM, overnight; left), nutrient-starved with EBSS (4 hr, middle), or untreated (right) using LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #13173 (red) and β-Actin (8H10D10) Mouse mAb #3700 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).Learn more about how we got this image
Gallery: LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #13173
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
- 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
- Methanol, 100%
- Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
- Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
- Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
B. Specimen Preparation - Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
- Aspirate liquid, then cover cells to a depth of 2–3 mm with ice-cold 100% methanol.
- Allow cells to fix for 15 minutes at -20°C.
- Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
- Proceed with Immunostaining (Section C).
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
- Block specimen in Blocking Buffer for 60 minutes.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 minutes each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
- For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.
posted December 2010
protocol id: 221
LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) recognizes endogenous levels of total LC3A and LC3B proteins.Species Reactivity: Human, Mouse, Rat Species predicted to react based on 100% sequence homology: Monkey, Xenopus, Bovine, Dog, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu44 of human LC3B protein (conserved in LC3A).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for direct immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated LC3A/B (D3U4C) XP® Rabbit mAb #12741.
Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation, but it has also been associated with a number of physiological processes including development, differentiation, neurodegenerative diseases, infection, and cancer (3). Autophagy marker Light Chain 3 (LC3) was originally identified as a subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) (4) and subsequently found to contain similarity to the yeast protein Apg8/Aut7/Cvt5 critical for autophagy (5). Three human LC3 isoforms (LC3A, LC3B, and LC3C) undergo post-translational modifications during autophagy (6-9). Cleavage of LC3 at the carboxy terminus immediately following synthesis yields the cytosolic LC3-I form. During autophagy, LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 that allows for LC3 to become associated with autophagic vesicles (6-10). The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form, LC3-II, have been used as indicators of autophagy (11).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. XP® is a trademark of Cell Signaling Technology, Inc. Alexa Fluor® is a registered trademark of Life Technologies Corporation. The Alexa Fluor® dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.