Flow cytometry was performed with the methods described previously with minor modifications.(19 (link)) We euthanized the CIA nestin-GFP transgenic mice 50 days after initial immunization, and collected marrow cells from the proximal epiphysis of tibias. Red blood cells were lysed by commercial ACK lysis buffer (Quality Biological, Inc., Gaithersburg, MD, USA). After centrifugation (200g, 5 min at room temperature), the cell pellet was resuspended and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich). The cells were washed with 0.1% BSA in PBS, and one million cells per milliliter were permeabilized in 0.1% Triton X-100 before blocking in 3% FACS buffer (PBS, 3% FBS, and 0.1% NaN3 sodium azide) for 30 min on ice. The cells were then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-GFP (abcam; 1:500) in Perm/Wash Buffer I (BD Biosciences, San Jose, CA, USA) for 1 hour at 4°C. After washing, cells were further incubated with peridinin chlorophyll protein (Per CP)-conjugated anti-CD45 (abcam; 1:500) in PBS containing 0.5% BSA for 30 min, and then analyzed with FACSCalibur flow cytometer and CellQuest software (BD Biosciences).
Fitc conjugated anti gfp
Manufactured by Abcam
Sourced in United States
FITC-conjugated anti-GFP is a fluorescently labeled antibody that binds to the green fluorescent protein (GFP). It can be used to detect and visualize GFP-tagged proteins in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (4 mentions)
Mouse anti β gal, by Promega (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Cellquest software, by BD (1 mentions)
Perm wash buffer 1, by BD (1 mentions)
Percp conjugated anti cd45, by Abcam (1 mentions)
Ack lysis buffer, by Quality Biological (1 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (1 mentions)
Axioplan 2 microscope, by Hamamatsu Photonics (1 mentions)
Alexa fluor 633 phalloidin, by Thermo Fisher Scientific (1 mentions)
Flag m2, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Polylysine coated slides, by Avantor (1 mentions)
Pannoramic midi 2 slide scanner, by 3DHISTECH (1 mentions)
Rabbit anti pkcζ c 20, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti expanded, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti apkcζ c20, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 546 647, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
7 protocols using fitc conjugated anti gfp
1
Immunological Profiling of Nestin-GFP Mice
2
Immunofluorescence Staining of Drosophila Tissues
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Ovaries and imaginal discs were dissected in PBS, fixed for 20 mins in 4% paraformaldehyde in PBS, washed for 30 minutes in PBS/0.1% Triton X-100 (PBST), and blocked for 15 minutes in 5% normal goat serum/PBST (PBST/NGS). Primary antibodies were diluted in PBST/NGS, and samples were incubated overnight at 4 °C.
The primary antibodies used were mouse anti-βgal (1:500; Promega, Madison, WI, USA), FITC-conjugated anti-GFP (1:400; Abcam, Cambridge, UK), mouse anti-Eya (1:250, DSHB), and mouse anti-Armadillo (1:100, Developmental Studies Hybrid Bank [DSHB], University of Iowa, Iowa City, IA, USA).
Secondary antibodies (all from Molecular Probes, Invitrogen, Carlsbad, CA, USA) were used at 1:500 for 2–4 hours prior to multiple washes in PBST and staining with DAPI at 1 μg/ml for 10–30 min prior to mounting on slides in Vectashield (Vector Laboratories, Burlingame, CA, USA).
The primary antibodies used were mouse anti-βgal (1:500; Promega, Madison, WI, USA), FITC-conjugated anti-GFP (1:400; Abcam, Cambridge, UK), mouse anti-Eya (1:250, DSHB), and mouse anti-Armadillo (1:100, Developmental Studies Hybrid Bank [DSHB], University of Iowa, Iowa City, IA, USA).
Secondary antibodies (all from Molecular Probes, Invitrogen, Carlsbad, CA, USA) were used at 1:500 for 2–4 hours prior to multiple washes in PBST and staining with DAPI at 1 μg/ml for 10–30 min prior to mounting on slides in Vectashield (Vector Laboratories, Burlingame, CA, USA).
3
Dissection and Immunohistochemistry of Drosophila Larval Neuromuscular Junction
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Wandering third instars were dissected in Ca2+-free saline and fixed in 4% paraformaldehyde for 30 min, except for Ena immunostaining, which required a fixation in a mix of 37% formaldehyde and 100% methanol for 10 min. The following primary antibodies were used for immunohistochemistry: anti-HRP, 1:1000 (123-095-021; Jackson ImmunoResearch, West Grove, PA, USA); FITC-conjugated anti-GFP, 1:1500 (ab6662; Abcam, Cambridge, MA, USA); and anti-Ena 1G6, 1:4 (a gift from F. M. Hoffmann, Madison, WI, USA). Antibodies obtained from the Developmental Studies Hybridoma Bank Iowa City, IA, USA include: anti-α-Spectrin 3A9, 1:30; anti-Cactus 3H12, 1:100; and anti-Dlg 4F3, 1:100. F-actin was visualized using Alexa Fluor 633 phalloidin (1:400; Invitrogen). Secondary antibodies conjugated with fluorophores FITC, Cy3 and AMCA (Jackson ImmunoResearch, West Grove, PA, USA) were used at a 1:200 dilution. RP3 and MN6/7b terminals of muscle 6 and 7 in the abdominal segment A2 of wandering third instar larvae were used for the quantification of all morphological parameters. This analysis was carried out using a Zeiss Axioplan2 microscope and a Hamamatsu ORCA wide-field digital camera as previously described (Loya et al., 2009 (link)). Bouton quantification was performed as previously described (Mosca and Schwarz, 2010 (link)).
4
PLA of Tat and FLAG M2 Proteins
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J-Lat 1C10 cells were washed with PBS and allowed to adhere to Polylysine coated slides (VWR, Radnor, PA, USA) marked with a hydrophobic barrier using A-PAP pen (Histolab, Gothenburg, Sweden). PLA was performed according to the manufacturer’s protocol (Sigma) with a few modifications: PLA plus and minus probes were diluted 1:20, amplification buffer (5×) was used at 10×. All washes were performed in PBS. Antibodies (1:1,000) were used against Tat (Abcam, Cambridge, United Kingdom), FLAG M2 (Sigma). Before DAPI staining and mounting with Duolink in Situ mounting media with DAPI (Sigma), FITC-conjugated anti-GFP (Abcam) was applied (1:500) for 1 h at ambient temperature protected from light. Slides were sealed with nail polish and stored at 4°C overnight before imaging. Slides were imaged using a Pannoramic Midi II slide scanner (3DHistech, Budapest, Hungary) and images were exported using the CaseViewer application. Images were analyzed with ImageJ (version 2.0.0-rc-69/1.52) and macros developed in house. Each RGB image was separated into separate channels. Based on the blue (DAPI) channel, nuclei were identified. In the red channel, spots (“maxima”) were detected with prominence thresholds 6–48.
5
Immunostaining of Drosophila Ovaries and Imaginal Discs
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Ovaries and imaginal discs were dissected in PBS, fixed for 20 min in 4% paraformaldehyde in PBS, washed for 30 min in PBS/0.1% Triton X-100 (PBST) and blocked for 15 min in 5% normal goat serum/PBST (PBST/NGS). Primary antibodies were diluted in PBST/NGS and samples were incubated overnight at 4°C.
Primary antibodies used were: mouse anti-Cut (1:100, DSHB), mouse anti-β-gal (Promega, 1:500), rabbit anti-expanded (1:200, a gift from A. Laughon, University of Wisconsin-Madison, USA), rabbit anti-PKCζ (C-20) (1:250, Santa Cruz), rat anti-Crumbs (1:300, a gift from U. Tepass, University of Toronto, Canada), mouse anti-Dlg (1:250, DSHB), rabbit anti-Kibra (1:200; Genevet et al., 2010 (link)), rabbit anti-Src pY418 (1:150, Life Tech), FITC-conjugated anti-GFP (1:400, Abcam), rabbit anti-βH-Spectrin (1:100, a gift from G. Thomas, Pennsylvania State University, USA), guinea pig anti-Merlin (1:100, a gift from R. Fehon, University of Chicago, IL, USA), mouse anti-eyes absent (1:250, DSHB) and mouse anti-Coracle (1:100).
Secondary antibodies (all from Molecular Probes or Invitrogen) were used at 1:500 for 2-4 h prior to multiple washes in PBST and staining with DAPI at 1 mg/ml for 10-30 min prior to mounting on slides in Vectashield (Vector labs).
Primary antibodies used were: mouse anti-Cut (1:100, DSHB), mouse anti-β-gal (Promega, 1:500), rabbit anti-expanded (1:200, a gift from A. Laughon, University of Wisconsin-Madison, USA), rabbit anti-PKCζ (C-20) (1:250, Santa Cruz), rat anti-Crumbs (1:300, a gift from U. Tepass, University of Toronto, Canada), mouse anti-Dlg (1:250, DSHB), rabbit anti-Kibra (1:200; Genevet et al., 2010 (link)), rabbit anti-Src pY418 (1:150, Life Tech), FITC-conjugated anti-GFP (1:400, Abcam), rabbit anti-βH-Spectrin (1:100, a gift from G. Thomas, Pennsylvania State University, USA), guinea pig anti-Merlin (1:100, a gift from R. Fehon, University of Chicago, IL, USA), mouse anti-eyes absent (1:250, DSHB) and mouse anti-Coracle (1:100).
Secondary antibodies (all from Molecular Probes or Invitrogen) were used at 1:500 for 2-4 h prior to multiple washes in PBST and staining with DAPI at 1 mg/ml for 10-30 min prior to mounting on slides in Vectashield (Vector labs).
6
Immunofluorescent Analysis of Ovarian Proteins
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Ovaries were dissected in PBS, fixed for 20 min in 4% paraformaldehyde (PFA) in PBS, washed for 45 min in PBS/0.1% Triton X‐100 (PBST) and blocked for 30 min in 5% normal goat serum (NGS)/PBST. Primary antibodies were diluted in NGS/PBST, and samples were incubated overnight at 4°C.
Primary antibodies used were FITC‐conjugated anti‐GFP (1:400; Abcam), rat anti‐Crb (1:300, a gift from U.Tepass), anti‐Ex (gift from R. Fehon), rabbit anti‐Kib (1:200, Genevet et al, 2010 (link)), rabbit anti‐aPKCζ (C20) (1:250 Santa Cruz), anti‐pWts (gift from K. Irvine), anti‐Rab5 (DSHB), anti‐Rab7 (DSHB), anti‐Rab11 (DSHB), anti‐alpha‐spec (DSHB). Secondary antibodies (goat Alexa Fluor 488, 546, 647; Invitrogen) were used at 1:500 for 2 h at RT, and samples were mounted in Vectashield (Vector Laboratories).
Primary antibodies used were FITC‐conjugated anti‐GFP (1:400; Abcam), rat anti‐Crb (1:300, a gift from U.Tepass), anti‐Ex (gift from R. Fehon), rabbit anti‐Kib (1:200, Genevet et al, 2010 (link)), rabbit anti‐aPKCζ (C20) (1:250 Santa Cruz), anti‐pWts (gift from K. Irvine), anti‐Rab5 (DSHB), anti‐Rab7 (DSHB), anti‐Rab11 (DSHB), anti‐alpha‐spec (DSHB). Secondary antibodies (goat Alexa Fluor 488, 546, 647; Invitrogen) were used at 1:500 for 2 h at RT, and samples were mounted in Vectashield (Vector Laboratories).
7
Immunofluorescence Imaging of Ovarian Tissues
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Ovaries were dissected in PBS, fixed for 20 min in 4% paraformaldehyde in PBS, washed for 30 min in PBS/0.1% Triton X-100 (PBT) and blocked for 15 min in 5% normal goat serum/PBT (PBT/NGS). Primary antibodies were diluted in PBT/NGS and samples were incubated overnight at 4 °C. Secondary antibodies were used for 2 h at room temperature and then mounted on slides in Vectashield (Vector Labs). Images were taken with a Leica SP5 confocal using 40x oil immersion objective and processed with Adobe Photoshop and ImageJ.
Primary antibodies used were: rat anti-Crumbs (1:200, E. Knust), mouse anti-Crumbs (Cq4) (1:10, DSHB), rat anti-Crb intra (1:500 M.Bhat), rabbit anti-Lgl (1:50, Santa Cruz), mouse anti-Dlg (1:250, DSHB) and FITC-conjugated anti-GFP (1:400, Abcam).
Secondary antibodies used were goat Alexa fluor 488, 546 or 647 (1:500, Invitrogen), Phalloidin (2.5:250, Life Technologies) to stain F-actin and DAPI (1 μg/ml, Life Technologies) to visualize nuclei.
Primary antibodies used were: rat anti-Crumbs (1:200, E. Knust), mouse anti-Crumbs (Cq4) (1:10, DSHB), rat anti-Crb intra (1:500 M.Bhat), rabbit anti-Lgl (1:50, Santa Cruz), mouse anti-Dlg (1:250, DSHB) and FITC-conjugated anti-GFP (1:400, Abcam).
Secondary antibodies used were goat Alexa fluor 488, 546 or 647 (1:500, Invitrogen), Phalloidin (2.5:250, Life Technologies) to stain F-actin and DAPI (1 μg/ml, Life Technologies) to visualize nuclei.
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