Imaginal discs were dissected and fixed in 4% formaldehyde/PBS for 18 min at 20 °C. Washing and blocking was performed in PBS + 0.1 Triton X-100 (PBT) and PBT + 5% normal goat serum, respectively. Discs were incubated with primary antibodies overnight at 4 °C: guinea pig anti-Spaghetti-squash 1 P (MRLC-1P) (1:400, gift from Robert Ward), rabbit anti-Cleaved Drosophila Dcp-1 (1:250) (Cell Signaling, 9578), mouse β-catenin (1:100, DSHB, N27A1) and rat anti-E-cadherin (1:100, DSHB, DCAD2). Discs were counterstained with DAPI (0.25 ng/μl, Sigma), Phalloidin (Alexa Fluor 488 and Alexa Fluor 647,1:100, Molecular Probes, or Phalloidin-TRITC, 1:400, Sigma). Secondary antibodies (coupled to Alexa Fluorophores, Molecular Probes) were incubated for 2 h at 20 °C. BrdU labeling on cultured discs was carried out as described70 (link). Discs were mounted using Molecular Probes Antifade Reagents. Samples were imaged using a Leica TCS SP5 confocal microscope equipped with HCX PL APO Lambda Blue 20x (NA 0.7) and HCX PL APO Lambda Blue 63x (NA 1.4) lenses.
Alexa fluorophore
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa fluorophores are a series of synthetic dyes developed for use in fluorescence-based applications. They are designed to provide bright, photostable fluorescence and are compatible with common fluorescence instrumentation. Alexa fluorophores are available in a range of spectral variants to suit various experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Triton x 100, by Merck Group (4 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (3 mentions)
Rabbit anti iba1, by Fujifilm (3 mentions)
Hoechst 33432, by Thermo Fisher Scientific (3 mentions)
Operetta cls system, by PerkinElmer (3 mentions)
Rabbit anti gfap, by Agilent Technologies (3 mentions)
Shandon cytospin 4, by Thermo Fisher Scientific (3 mentions)
Prolong gold, by Thermo Fisher Scientific (3 mentions)
Lsm 800, by Zeiss (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti chicken 488, by Thermo Fisher Scientific (2 mentions)
Anti rat 488 and 568, by Thermo Fisher Scientific (2 mentions)
Anti rabbit 568, by Thermo Fisher Scientific (2 mentions)
Chicken anti gfp, by Aves Labs (2 mentions)
Rabbit anti rfp, by Rockland Immunochemicals (2 mentions)
Phalloidin alexa fluor 488 647, by Thermo Fisher Scientific (2 mentions)
Tcs sp8 microscope, by Leica camera (2 mentions)
65 protocols using alexa fluorophore
1
Immunohistochemical Analysis of Drosophila Imaginal Discs
2
Immunofluorescence Staining and Imaging
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Cells were fixed in 4% PFA and incubated for 30 min in blocking buffer (PBS and 0.3% Triton X-100 and 5% horse serum). Primary antibodies (table S7) were diluted in antibody solution (PBS and 0.1% Triton X-100 and 1% horse serum) and applied overnight at RT. After three washes in PBS, secondary antibodies conjugated to Alexa fluorophores (Molecular Probes, Eugene, OR, USA) were diluted at 1:1000 in antibody solution and applied for 1 hour at RT. Cells were washed in PBS followed by nuclear staining by DAPI (Sigma-Aldrich) for 10 min. Cells were washed three more times and mounted by VECTASHIELD Mounting Medium-Vector Laboratories. Confocal image acquisition was performed using Olympus FV1200 and SP8 Leica DLS.
3
Immunostaining of Pluripotency Markers
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Cells were grown on glass cover slips, fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS). Antibodies used were: Cdx2 (Biogenex CDX2–88) at 1:400, Elf5 (Santa Cruz Biotechnology N-20 sc-9645) 1:200, His-probe (Santa Cruz Biotechnology) 1:100, FLAG (M2, Sigma F1804) 1:400, Nanog (Abcam ab19857) 1:400, Oct4 (Abcam ab19857) 1:400, Parp1 (Santa Cruz Biotechnology sc-74469x 1:200 and Abcam ab18376 1:100, both giving identical results), Parp7 (Abcam ab170817) 1:100 and Stella (Abcam ab19878) 1:100. Secondary antibodies were Alexa fluorophores (Molecular Probes) at 1:400, and DNA was visualized with DAPI or bis-benzimide. Photographs were taken on an Olympus BX41 epifluorescence microscope and a Zeiss 510 Meta confocal microscope, and analysed with Volocity software (Improvision). Cells (n > 1000) were classified as positive or negative for each factor analysed and data compared using a Chi-squared test (*P < 0.05, **P < 0.01, ***P < 0.001).
4
Immunofluorescence and FISH Protocol
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Cells grown on coverslips were fixed with 2% paraformaldehyde and permeabilized with 0.5% NP-40. Immunofluorescence staining was carried out by incubating with an anti-BLM antibody (Santa Cruz Biotechnology, sc-7790, 1:150), an anti-RPA34 antibody (GeneTex, clone 9H8, 1:500), an anti-PICH antibody (Abnova, H54821-B01P, 1:500), or an anti-TRF1 antibody (GeneTex, clone 4E4, 1:500), followed by secondary antibody conjugated with respective Alexa Fluorophores (Molecular Probes, 1:500). The cells were fixed again with 4% paraformaldehyde and dehydrated by successive incubation in 70, 95 and 100% ethanol before subjected to FISH analysis. PNA probes for FISH analysis were TMR-, Cy5- or Alexa488-OO-5′-(CCCTAA)3-3′ (telomeric sequence). DNA was stained by 0.1 μg ml−1 DAPI. Coverslips were mounted onto glass slides in Prolong Gold Antifade Reagent (Invitrogen). Images were acquired with a Nikon Ti-U microscope using a × 100 objective and collected as a stack of 0.2-μm increments in the z axis. Image deconvolution was conducted using the AutoQuant X3 software. Unless otherwise noted, images were viewed as a single section on the z axis.
5
Flow Cytometry and Microscopy Antibodies
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The antibody clones used for flow cytometry were CD8 (BioLegend, 53-6.7), CD62L (eBioscience, MEL-14), CD44 (eBioscience, IM7) CD107a/LAMP1 (BD Biosciences, H4A3) and NKG7 (Cell Signaling Technology, E6S2A); for microscopy were EEA1 (Cell Signaling Technology, E9Q6G), Rab27 (Cell Signaling Technology, E907E), CD107a/LAMP1 (BioLegend, H4A3) Granzyme B (BD Biosciences, GB11) and rabbit and mouse anti-tubulin (Rockland). Secondary antibodies conjugated to Alexa Fluorophores, ProLong™ Gold Antifade Mountant with DAPI, CellTrace™ Violet and CFSE dyes were purchased from Molecular Probes (Thermo Fisher). Calcein-AM and LysoTracker™ were purchased from Invitrogen (Thermo Fisher).
6
Immunofluorescence and Immunohistochemistry of Cryosections
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Cryosections (7 μm thick) were labelled without prior fixation using primary antibodies diluted in PBS 0.1% Tween-20. Immunofluorescence was visualised with species-specific secondary antibodies directly linked to Alexa-fluorophores (Molecular Probes, Eugene, OR, USA). Nuclei were visualised using DAPI (Molecular Probes, Eugene, OR, USA) diluted in PBS 0.1% Tween-20. Immunohistochemistry was performed using Novo-Link peroxidase detection kit according to manufacturer’s instructions (Novo-Link peroxidase detection kit, Leica Microsystems, Wetzlar, Germany). We used mouse monoclonal antibody against MHC Class I (Dako, Glostrup Denmark, Cat. M0736, 1:1000) and rabbit polyclonal antibody against active caspase-3 (Pharmingen, San Diego, CA, USA, Cat. 559565, 1:400). Non-specific labelling was assessed using sections incubated without primary antibodies. Immunofluorescence was visualised under a conventional epifluorescence microscope (Leica DM5000B). Images were acquired with Leica Imaging Suite (Leica Microsystems, Wetzlar, Germany) and were quantified with ImageJ software (http://rsbweb.nih.gov/ij/ ).
7
Immunofluorescence Staining Protocol for Cellular Organelles
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Immunofluorescence was performed in cells grown on glass slides (Thermo Fisher FALC354108). HCT116 cells were fixed for 15 min in 4% paraformaldehyde, washed twice for 5 min in PBS, and permeabilized in 0.1 % Triton X‐100 in PBS for 10 min at RT. After two more washes for 5 min in PBS, cells were blocked in 10% horse serum for 10 min at RT and subsequently treated with primary antibodies in 5% horse serum for 1 h. Primary antibodies used were Lamp1 (553792) and p62 (610832) from BD Biosciences (San Jose, CA), and LC3b (L8918) from Sigma (Taufkirchen, Germany).
Afterward, cells were washed three times for 5 min in PBS and incubated for 1 h with secondary antibodies labeled with Alexa fluorophores (1:1,000) and Alexa‐488‐phalloidin (1:200) from Thermo Fisher Scientific (Waltham, MA) at RT. Subsequently, cells were washed twice with PBS and stained with Dapi, and then mounted onto glass slides with 0.1 g/ml Mowiol.
For cholesterol staining, the Cholesterol Cell‐Based Detection Assay Kit (Cayman Chemical Item No. 10009779) was used following the manufacturer’s instructions.
Afterward, cells were washed three times for 5 min in PBS and incubated for 1 h with secondary antibodies labeled with Alexa fluorophores (1:1,000) and Alexa‐488‐phalloidin (1:200) from Thermo Fisher Scientific (Waltham, MA) at RT. Subsequently, cells were washed twice with PBS and stained with Dapi, and then mounted onto glass slides with 0.1 g/ml Mowiol.
For cholesterol staining, the Cholesterol Cell‐Based Detection Assay Kit (Cayman Chemical Item No. 10009779) was used following the manufacturer’s instructions.
8
Immunofluorescence Antibody Protocol
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Mouse monoclonal S9.6, rabbit polyclonal αCID and rat polyclonal αHP1a are described in34 (link), 61 (link), 62 (link). Rabbit polyclonal αdH1 was kindly provided by Dr Kadonaga and is described in ref. 11 (link). All other antibodies used in these experiments were commercially available: monoclonal mouse αγH2Av (DSHB, UNC93-5.2.1) or rabbit αγH2Av (Rockland, 600-401-914), rabbit polyclonal αH2Av (Active Motif, 39716), rabbit polyclonal αH4 (Abcam Ab10158), rabbit polyclonal αCaspase 3 (Cell Signalling, 9661S), mouse polyclonal α−βtubulin (EMD-Millipore, MAb3408), rat monoclonal αBrDU, (AbD Serotec, OBT0030G) and mouse monoclonal αBrDU (Becton Dickinson, 347580). Commercial secondary antibodies used for immunofluorescence were either coupled to cyanines (Jackson) or to Alexa fluorophores (ThermoFisher), and to horseradish peroxidase for WB (Jackson).
9
Intratumoral MET Protein Imaging in Mice
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All mouse experiments were conducted in accordance with the guidelines of and approved by the Regeneron Institutional Animal Care and Use Committee. Six-week-old female SCID mice (Jackson LAb, 001803) were injected with 150 μL EBC1 cell suspension in matrigel (Corning) at 33 million cells per milliliter. A total of 10 days after implantation tumors measuring approximately 200 mm3 were injected with 5 μg/mL METxMET Ab or METxMET-VC-biosensor. A total of 24 hours after injection, tumors were harvested, fixed with 30% sucrose-DPBS (3 hours, 4°C) followed by 4% PFA-DPBS (16 hours, 4°C) with gentle rotation. Fixed tumors were embedded in optimal cutting temperature compound (Tissue-Tek), flash frozen, cut in think sections and processed for immunofluorescence staining and confocal imaging.
Tissue sections were permeabilized with 0.1% Triton X-100 (Sigma)-DPBS (10 minutes, room temperature), blocked with 1% BSA-DPBS (60 minutes, room temperature) and incubated with primary antibodies previously labeled with Alexa fluorophores (Thermo Fisher Scientific) in 1% BSA-DPBS (60 minutes, room temperature). Samples were washed twice with 1% BSA-DPBS, once with DPBS (5 minutes, room temperature), covered with mounting medium (biotium) and sealed before imaging.
Tissue sections were permeabilized with 0.1% Triton X-100 (Sigma)-DPBS (10 minutes, room temperature), blocked with 1% BSA-DPBS (60 minutes, room temperature) and incubated with primary antibodies previously labeled with Alexa fluorophores (Thermo Fisher Scientific) in 1% BSA-DPBS (60 minutes, room temperature). Samples were washed twice with 1% BSA-DPBS, once with DPBS (5 minutes, room temperature), covered with mounting medium (biotium) and sealed before imaging.
10
Immunofluorescence Staining of Ciliary Proteins
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For the antibodies Ac-Tu, ARL13B, ARL3, INPP5E, γ-tubulin, SMO, and AC3), cells were fixed with 4% PFA in phosphate-buffered saline (PBS) prewarmed to 37°C on the benchtop for 15 min. Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. For Ac-Tu, ARL13B, and SMO, cells were blocked with 1% bovine serum albumin (BSA; Sigma #A3059) in PBS for 1 h. Primary antibodies were diluted in blocking solution and applied to cells at 4°C overnight. Cells were washed 4 × 5 min with PBS before incubation with secondary antibodies (1:1000; Alexa fluorophores; ThermoFisher) in blocking solution for 1 h at room temperature. Cells were washed 4 × 5 min with PBS before being mounted onto slides with MOWIOL. For ARL3, INPP5E, and AC3, a blocking solution of 10% FBS in PBS was used in place of 1% BSA.
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