Immunofluorescence was performed as previously described45 (link). Hemithoraces were dissected and fixed for 20 minutes in PBS with 4% paraformaldehyde and 0.1% Triton X-100. After washing, samples were incubated overnight at 4ºC with an antibody against poly-ubiquitinated proteins, mouse mAb FK2 (Enzo), and anti-P62 rabbit ab178440 (Abcam) at 1:200 dilutions, then washed thoroughly and incubated with secondary anti-mouse AlexaFluor-488 (1:250), anti-rabbit AlexaFluor-555 (1:250), and phalloidin AlexaFluor-647 (1:150) (all Invitrogen). Samples were rinsed three times in PBS+0.1% triton X-100 for 10 minutes at room temperature, then mounted in Prolong Gold (Invitrogen) and imaged via confocal microscopy using 20X dry objective with 0.5 numerical aperture (NA) (LSM-800 Zeiss, standard laser lines: 405, 488, 561, 640 nm). Identical laser power settings and photomultiplier values were used for imaging between conditions. For quantification of protein aggregates of hemithoraces, the size and area of protein aggregates was measured using ImageJ particle counter software (ImageJ version 2.0.0-rc-69/1.52p).46 (link) Statistical analysis was conducted using a two-tailed, unpaired Student’s t-test or ANOVA followed by Tukey’s multiple comparisons (n≥9 thoraces per condition).
Ab178440
Manufactured by Abcam
Sourced in United Kingdom
Ab178440 is an antibody used for Western blotting applications. It is a rabbit polyclonal antibody that recognizes a specific target protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Anti rabbit alexafluor 555, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Anti lc3b, by Merck Group (1 mentions)
Anti p62 nbp1 48320, by Novus Biologicals (1 mentions)
Anti phospho tau at8, by Thermo Fisher Scientific (1 mentions)
Ir dye 700 800, by Rockland Immunochemicals (1 mentions)
Goat anti rabbit cy5, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 555 secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Ab14748, by Cell Signaling Technology (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
P s65 ub, by Cell Signaling Technology (1 mentions)
Mowiol 4 88, by Merck Group (1 mentions)
Anti mouse cytm3, by Jackson ImmunoResearch (1 mentions)
9 protocols using ab178440
1
Immunofluorescence Quantification of Protein Aggregates
2
Antibody Usage for Autophagy Analysis
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The following commercially available antibodies were used: anti-GABARAP for Drosophila Atg8a (PM037, MBL), anti-Ref(2)P (ab178440, Abcam), anti-Tau 5A6 (5A6, DSHB), anti-Phospho-Tau AT8 (MN1020, Thermo), anti-cleaved caspase 3 (9661, Cell Signaling), anti-LC3B (L7543, Sigma), anti-p62 (NBP1-48320, Novus Biologicals), anti-GFP (G5144, Invitrogen), anti-Actin (A1978, Sigma), Cy5-conjugated anti-HRP (123175021, Jackson ImmunoLab), and secondary antibodies conjugated to Alexa 488/568/647 (Thermo Fisher Scientific), or near infrared (IR) dye 700/800 (Rockland).
3
Immunostaining of Muscle Tissue
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Adult abdominal and larval muscles were dissected in saline and fixed with 4% paraformaldehyde for 15 min or Bouin's solution for 10 min. The dissected muscles were washed with 2% PBS-Triton X-100 and incubated with rabbit polyclonal antibody Ref(2)p/p62 (rabbit pAb, Abcam, ab178440) at a 1:500 dilution, TDP-43 rabbit polyclonal antibody (pAb, Proteintech, 10782-2-Ap) at a 1:500 dilution or Lamin C mouse monoclonal antibody (mAb, Developmental Studies Hybridoma Bank, LC28.26) at a 1:100 dilution overnight at 4°C on a nutator. Subsequently, the muscles were washed three times with 2% PBS-Triton X-100 and incubated overnight at 4°C with either goat anti-rabbit-Cy5 or goat anti-mouse-Alexa 555 secondary antibodies (Invitrogen) at a 1:500 dilution. Samples were mounted on a glass slide with Vectashield antifade mounting medium and imaged using a Leica DMi8 microscope with a 100× objective (1.4NA) and THUNDER imager.
4
Immunostaining of Drosophila Neuronal and Muscle Tissues
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Drosophila brains were dissected from 30-day-old flies and immunostained with anti-tyrosine hydroxylase as described previously [41 (link)]. Tyrosine hydroxylase-positive neurons were counted under blinded conditions. For immunostaining of adult flight muscles and larval brains, animals were dissected in PBS 1X and fixed in 4% FA/PBS for 30 min at room temperature, permeabilized in 0.3% Triton X-100 for 30 min, and blocked with 1% BSA in 0.3% Triton X-100 PBS for 1 h at RT. Tissues were then incubated on a shaker with the appropriate primary antibody: ATP5A antibody (Abcam, ab14748, 1:500), pUb (pS65-Ub, Cell Signalling Technologies, 62,802 S, 1:250) and/or ref(2)P/p62 (Abcam, ab178440, 1:1000), diluted in 1% BSA in 0.3% Triton X-100 PBS overnight at 4 °C. Next day, samples were rinsed 3 times 10 min with 0.3% Triton X-100 in PBS, and incubated with the appropriate fluorescent secondary antibodies overnight at 4 °C. The tissues were washed 3 times in 0.3% Triton X-100 in PBS followed by a last wash on PBS and mounted on slides using Prolong Diamond Antifade mounting medium (Thermo Fisher Scientific) and imaged next day. Tissues were imaged with a Zeiss LSM880 confocal microscope (Carl Zeiss MicroImaging) equipped with a Nikon Plan-Apochromat 63x/1.4 NA oil immersion objective. Images were analyses using FIJI (Image J).
5
Immunofluorescence Imaging of Drosophila Larval Nervous System
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Third instar larvae, which were raised at 28 °C, were harvested and dissected, as previously described [41 (link)]. Afterwards, they were incubated with the primary antibodies rabbit anti-Ref(2)p (1:200, ab178440, Abcam plc, Cambridge, UK), mouse anti-Elav (1:100, 9F8A9, DSHB, Iowa City, IA, USA), and mouse anti-Repo (1:100, 8D12, DSHB, Iowa City, IA, USA) over-night at +4 °C. After washing, the larvae were incubated in the secondary antibody anti-Rabbit CyTM5 (1:500, Cat# 111-175-144) or anti-Mouse CyTM3 (1:500, Cat# 115-165-003) (Jackson ImmunoResearch Europe Ltd., Cambridge, UK) for 1 h at RT then washed and mounted on glass slides using Mowiol® 4-88 (Cat# 324590, Sigma-Aldrich, St. Louis, MO, USA).
6
Western Blot Analysis of Fly Head Proteins
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Five fly heads were homogenized in SDS sample buffer (LC2676, Invitrogen). Lysates were centrifuged. The resulting supernatant was collected, and proteins were resolved on 15% Tris-tricine gels. The protein samples were transferred to nitrocellulose membranes with 0.22 µm pore size (Pall Corporation). The membranes were blocked with 5% nonfat dry milk and incubated with anti-β-Amyloid (1:2000, #8243 CST), anti-GABARAP (1:2000, #13733 CST), anti-Ubiquitin (1:2000, #3936 CST), anti-Ref2P (1:2000, ab178440 Abcam) and anti-GAPDH (1:50000, GTX100118 GeneTex) primary antibodies in TBST at 4°C overnight. The rabbit horseradish peroxidase-conjugated secondary antibody was used, and the signal was detected by chemiluminescence. The signal intensity was quantified by Image J software.
7
Western Blot Analysis of Cellular Proteins
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Protein extracts were prepared in Laemmli loading buffer containing 2.5% beta-mercaptoethanol (except for experiment in non-reducing condition where no beta-mercaptoethanol was added) and heated for 5 min at 95°C before separation of 20 μg total proteins on 8 or 12% SDS-PAGE gels. Separated proteins were transferred onto nitrocellulose or PVDF membranes. The membranes were blocked in TBS (Tris-buffered saline; 50 mM Tris-Cl, pH 7.6, 150 mM NaCl), 0.1% Tween-20, 5% non-fat milk. The following antibodies were used: anti-GFP (Santa Cruz sc-9996, 1:1,000), anti-GABARAP/Atg8a (Cell Signaling Technology No. 13733, 1:2,000), anti-Ref(2)P (Abcam ab178440, 1:1,000), anti-β actin (Abcam ab8227, 1:2,000), anti-α tubulin (Sigma-Aldrich T5168, 1:40,000), HRP-coupled secondary antibodies anti-rabbit and anti-mouse (Thermo Scientific No. 31460 and 31450, 1:10,000). Signals were developed using the ECL detection reagents (Amersham, RPN2209).
8
Immunofluorescence Microscopy of Drosophila IFMs
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IFMs were prepared from the adult thoraxes dissected in relaxing buffer (0.1 M KCl, 20 mM Tris-HCl, pH = 7.2, 1 mM MgCl2, 1 mM EDTA) [34 (link)]. The specimens were fixed in 4% paraformaldehyde for 30 min. After washing with 0.1% PBST (1× PBS, 0.1% TritonX-100) and subsequent blocking in 10% normal goat serum, the primary antibody diluted with the blocking solution was added and incubated at 4 °C overnight. The following primary antibodies were used: anti-Atg8 antibody (#ab109364, Abcam, Cambridge, UK) (dilution 1/400), anti-Ref(2)P antibody (#ab178440, Abcam) (1/500), anti-ATP5A antibody (#ab14748, Abcam) (1/400), and anti-Cleaved Drosophila Dcp-1 antibody (#9578, Cell Signaling Technology, Inc., Danvers, MA, USA) (1/100).
After washing IFM samples with 0.1% PBST, Alexa fluorescence dye-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) were incubated with the samples for 2 h. For the visualization of F-actin, Alexa Fluor 488-conjugated phalloidin (#A12379, Invitrogen) was added simultaneously. After washing with 0.1% PBST several times, the specimens were embedded with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and observed using a laser scanning confocal microscope (FV10i, Olympus, Tokyo, Japan). Images were acquired at 512 × 512 pixel size. For image processing, FV10-ASW 4.2 Viewer (Olympus) was used.
After washing IFM samples with 0.1% PBST, Alexa fluorescence dye-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) were incubated with the samples for 2 h. For the visualization of F-actin, Alexa Fluor 488-conjugated phalloidin (#A12379, Invitrogen) was added simultaneously. After washing with 0.1% PBST several times, the specimens were embedded with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and observed using a laser scanning confocal microscope (FV10i, Olympus, Tokyo, Japan). Images were acquired at 512 × 512 pixel size. For image processing, FV10-ASW 4.2 Viewer (Olympus) was used.
9
Immunohistochemistry of Drosophila Brains
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As with LysoTracker, flies were anesthetized on ice and immobilized in dissection collars. The proboscis was removed under cold dissection saline and then freshly diluted 4% paraformaldehyde (PFA) was added to the dissection collar (32% EM grade PFA (EMS)). Brains were fixed for 25 minutes; after 5 minutes of immersion in 4% PFA, the remaining head cuticle and surrounding fat was gently removed. Post-fixation, brains were washed three times in 1X PBS before completing the dissection in collars and removing brains into Terasaki wells with 0.5%PBSTx (TritonX-100 0.5% in 1X PBS; for GlcCer and Nile Red staining, 0.05% PBSTx was used to better preserve lipids). Brains were permeabilized for 30 mins then transferred to blocking solution (10%NGS in 0.5%PBSTx) for 40 minutes before adding primary antibodies in 0.5%PBSTx+10%NGS (1:10 CSP, 1:200 anti-FK2 polyubiquitin, 1:500 anti-p62 (Abcam ab178440), 1:20 nc82, 1:50 anti-PDF, 1:2000 anti-Gba1b, 1:10,000 anti-GFP (Abcam ab13970), 1:500 anti-GlcCer (RAS_0010 anti-rabbit Glycobiotech) for 24-48 hours at 4°C while rocking gently. Brains were then washed three times in 0.5%PBSTx before transfer to appropriate secondaries (1:500, Thermo Fisher Scientific) and rocked at 4°C overnight. Brains were washed three times in 0.5%PBSTx before placing in 70% glycerol for clearing, then mounted and imaged in Vectashield.
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