Immunofluorescence was performed as previously detailed12 (link) using anti-Vav1 monoclonal antibodies (mAbs) and secondary AlexaFluor-647 anti-mouse IgG, anti-Alexa fluor-546 phalloidin staining for actin cytoskeleton (Molecular Probes, USA; #A22283), anti-vimentin (Progen GP53; Germany), and Cy3 donkey anti-guinea pig (Jackson Laboratories, ME#705-165-148). Nuclear staining was detected by Dapi.
Cy3 donkey anti guinea pig
Manufactured by Jackson ImmunoResearch
Sourced in Germany, Denmark, United States, Cameroon
Cy3 donkey anti-guinea pig is a secondary antibody conjugated with the fluorescent dye Cy3. It is designed to detect and visualize guinea pig primary antibodies in various immunoassays and immunohistochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (3 mentions)
Cy2 donkey anti rabbit, by Jackson ImmunoResearch (3 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (2 mentions)
Donkey anti mouse cy5, by Jackson ImmunoResearch (2 mentions)
Donkey anti rabbit fitc, by Jackson ImmunoResearch (2 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit cy3, by Jackson ImmunoResearch (2 mentions)
Mouse α mapk yt, by Merck Group (2 mentions)
Lsm 800, by Zeiss (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Ma1 065, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Mouse anti pdf, by Developmental Studies Hybridoma Bank (1 mentions)
Donkey anti rat cy3, by Jackson ImmunoResearch (1 mentions)
Fluoview 1000 confocal microscope, by Olympus (1 mentions)
Mouse anti ha, by BioLegend (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Formalin, by Merck Group (1 mentions)
Guinea pig anti swine insulin, by Agilent Technologies (1 mentions)
12 protocols using cy3 donkey anti guinea pig
1
Immunofluorescence Staining Protocols
2
Immunostaining of Synaptic Proteins in INS-1 832/13 Cells
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INS‐1 832/13 cells were cultured in μ‐Slide (chambered coverslip) with 8 wells (LAB‐TEK, 154534) 1 day prior to immunostaining. The cells were first washed twice with PBS (Hyclone) and fixed with 3% PFA‐K‐PIPES and 3% PFA‐Na2BO4 for 5 and 10 min, respectively, followed by permeabilization with 0.1% Triton‐X 100 for 30 min at room temperature. The cells were incubated with the blocking solution containing 5% normal donkey serum (Jackson immunoresearch) in PBS for 30 min. Primary antibodies against Syt11 (1:50, # E‐AB‐10622, Elabscience), Syt13 (1:100, # ab‐154 695, Abcam), insulin (1:400, #16049, Progen), Syntaxin1a (1:100, #VAP‐SV064‐E, ENZO), or Clathrin (1:100, #MA1‐065, Invitrogen) were diluted in blocking solution and incubated overnight at 4°C. Immunoreactivity was quantified using fluorescently labeled secondary antibodies either Alexa Fluor 488, donkey anti‐rabbit (1:300, Jackson ImmunoResearch), or Cy 3, donkey anti‐guinea pig (1:300, Jackson ImmunoResearch). The Confocal images were acquired using a Zeiss LSM 800 on a 63× oil immersion objective. The fluorescent intensity and Manders colocalization coefficient were analyzed with software ZEN (2.6 Blue edition).
3
Immunolabeling of Drosophila Brains
4
Immunostaining of C. elegans Gonads
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Immunostaining of dissected gonads was carried out as described (Colaiacovo et al., 2003 ; Saito et al., 2009 ). Worms were permeabilized on Superfrost+slides for 2 min with methanol at −20° and fixed for 30 min in 4% paraformaldehyde in PBS. After blocking with 1% BSA in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature, slides were incubated with primary antibody for 1 h at room temperature. After incubation with fluorescent secondary antibody 1 h at room temperature, slides were DAPI‐stained for 10 min at 500 ng/ml, destained 1 h in PBST, and mounted with Vectashield (Vector Laboratories). The primary antibodies used were as follows: rabbit α‐LAB‐1 (1:200, (de Carvalho et al., 2008 )), rabbit α‐RAD‐51 (1:10,000, SDIX), mouse α‐MAPK‐YT (1:500, M8159; Sigma), rabbit α‐SYP‐2 (1:200, a kind gift from S. Smolikove), rabbit α‐pH3 (D5692, 1:1000; Sigma), and guinea pig α‐HTP‐3 (1:200, (Goodyer et al., 2008 )). The secondary antibodies used were Cy2‐donkey anti‐rabbit, Cy3‐donkey anti‐guinea pig, Cy3‐goat anti‐rabbit, and Cy3‐goat anti‐mouse (all used at 1:500 dilution; Jackson ImmunoResearch Laboratories).
5
Immunolabeling of Drosophila Adult Brains
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Adult fly brains were dissected in cold phosphate-buffered saline with 0.1% Triton-X (PBST) and fixed in 4% formaldehyde for 20–35 min. Brains were rinsed 3 X 15 min with PBST, blocked for 60 min in 5% normal donkey serum in PBST (NDST), and incubated for 24 hrs at RT in primary antibody diluted in NDST. Brains were then rinsed 3 X 15 min in PBST, incubated for 24 hrs in secondary antibody diluted in NDST, rinsed 3 X 15 min in PBST, cleared for 5 min in 50% glycerol in PBST, and mounted in Vectashield. Primary antibodies were as follows: rabbit anti-GFP 1:1000 (Molecular Probes A-11122), rat anti-RFP 1:1000 (Chromotek 5F8), mouse anti-HA 1:250 (BioLegend 901501), rabbit anti-SIFa 1:4000 (gift of J. Veenstra), and guinea pig anti-PERIOD 1:1000 (UPR 1140; gift of A. Sehgal). Secondary antibodies were as follows: FITC donkey anti-rabbit 1:1000 (Jackson 711-095-152), Cy3 donkey anti-rat 1:1000 (Jackson 712-16-150), Cy5 donkey anti-mouse 1:1000 (Jackson 715-175-151) and Cy3 donkey anti-guinea pig 1:1000 (Jackson 706-165-148). Immunolabeled brains were visualized with a Fluoview 1000 confocal microscope (Olympus). Trans-Tango flies were raised at 18°C and dissected ~2 weeks post-eclosion to maximize signal intensity [25 (link)].
6
Immunostaining Dissected Gonads in Worms
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Immunostaining of dissected gonads was carried out as described (Colaiacovo et al. 2003; Saito et al. 2009) . Worms were permeabilized on Superfrost+ slides for 2 min with methanol at -20° and fixed for 30 min in 4% paraformaldehyde in PBS. After blocking with 1% BSA in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature, slides were incubated with primary antibody for 1 h at room temperature.
After incubation with fluorescent secondary antibody 1 h at room temperature, slides were DAPI stained for 10 min at 500 ng/ml, destained 1 h in PBST, and mounted with Vectashield (Vector Laboratories). The primary antibodies used were as follow: rabbit α-LAB-1 (1:200, (de Carvalho et al. 2008 )), rabbit α-RAD-51 (1:10,000, SDIX), mouse α-MAPK-YT (1:500, M8159; Sigma), rabbit α-SYP-2 (1:200, a kind gift from S. Smolikove), rabbit α-pH3 (D5692, 1:1000; Sigma), and guinea pig α-HTP-3 (1:200, (Goodyer et al. 2008) ). The secondary antibodies used were Cy2-donkey anti-rabbit, Cy3-donkey anti-guinea pig, Cy3-goat anti-rabbit, Cy3-goat anti-mouse (all used at 1:500 dilution; Jackson ImmunoResearch Laboratories).
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted October 29, 2020. ; https://doi.org/10.1101/2020.10.29.360693 doi: bioRxiv preprint
After incubation with fluorescent secondary antibody 1 h at room temperature, slides were DAPI stained for 10 min at 500 ng/ml, destained 1 h in PBST, and mounted with Vectashield (Vector Laboratories). The primary antibodies used were as follow: rabbit α-LAB-1 (1:200, (de Carvalho et al. 2008 )), rabbit α-RAD-51 (1:10,000, SDIX), mouse α-MAPK-YT (1:500, M8159; Sigma), rabbit α-SYP-2 (1:200, a kind gift from S. Smolikove), rabbit α-pH3 (D5692, 1:1000; Sigma), and guinea pig α-HTP-3 (1:200, (Goodyer et al. 2008) ). The secondary antibodies used were Cy2-donkey anti-rabbit, Cy3-donkey anti-guinea pig, Cy3-goat anti-rabbit, Cy3-goat anti-mouse (all used at 1:500 dilution; Jackson ImmunoResearch Laboratories).
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted October 29, 2020. ; https://doi.org/10.1101/2020.10.29.360693 doi: bioRxiv preprint
7
Histological Analysis of Islet Grafts
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Kidneys containing islet grafts were fixed in 10% formalin (Sigma-Aldrich). A cut was made through the center of the graft 24 h later, and the kidneys were then embedded in paraffin. For histological examination, hematoxylin and eosin (H&E; Dako, Carpinteria, CA, USA) staining and immunofluorescence staining were performed. Insulin immunostaining was performed with guinea pig anti-swine insulin (1:200; Dako Cytomation, Glostrup, Denmark) , detected by Cy3 donkey anti-guinea pig (1:200; Jackson ImmunoResearch, West Grove, PA, USA). Nuclei were stained with 4¢,6-diamidino-2phenylindole (DAPI) (1 µg/ml; Sigma-Aldrich). Immunofluorescence was detected with an Olympus BX60 microscope (Olympus UK Ltd., London, UK).
8
Circadian Clock Protein Localization
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Brains from w1118; + / + ; 3xHA-Nipped-A, w1118, UAS-dicer; 3 × HA-Nipped-A/ + ; Nipped-A RNAi3/ + and w1118, UAS-dicer; 3 × HA-Nipped-A/tim-Gal4; Nipped-A RNAi3/ + flies were collected at ZT0, dissected, fixed, immunostained and imaged by confocal microscopy as described62 (link). The primary antibodies used were anti-CLK GP5062 (link) at a 1:3000 dilution, preabsorbed rabbit anti-PER antibody (a gift from Michael Rosbash) at a 1:20,000 dilution and mouse anti-HA antibody (Sigma #H9658) at a 1:1000 dilution. Secondary antibodies used were donkey anti-rabbit Alexa 488 (Jackson ImmunoResearch #711-545-152), donkey anti-mouse Alexa 488 (Jackson ImmunoResearch #715-545-150), donkey anti-Guinea pig Cy3 (Jackson ImmunoResearch #706-165-148) and donkey anti-mouse Cy3 (Jackson ImmunoResearch #715-165-150), all at a 1:200 dilution. Confocal stacks were imaged using an Olympus FV1000 confocal microscope equipped with 20 × 0.85 NA and 100 × 1.40 NA oil-immersion objectives as described23 (link).
9
Immunohistochemical Staining of Brain Sections
10
Perfusion and Immunohistochemistry of Rodent Brains
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Adult animals were anesthetized with a ketamine (250 mg/kg) and xylazine (25 mg/kg) solution (i.p.) and transcardially perfused with 4% paraformaldehyde in PBS (1 ml/g). E18.5 embryos were excised after deep anesthesia of pregnant mothers; embryos' brains were extracted and immersed in fixative. After anesthesia in isoflurane, P0–P2 brains were extracted and immersed in fixative, whereas P3–P8 pups were perfused with paraformaldehyde before brain extraction. Brains were postfixed overnight, washed in PBS, then placed in 30% sucrose in PBS before freezing in liquid nitrogen and kept at –20°C for long‐term storage.
Coronal or horizontal brain sections were prepared and processed for immunohistochemistry as described previously (Picardo et al.,2011 ). Various primary antibodies were used (Table 2 ). Secondary antibodies (dilution 1:500) used were donkey anti‐chicken DyLight 488, donkey anti‐rabbit DyLight 549, donkey anti‐guinea pig Cy3, donkey anti‐mouse DyLight 549, and donkey anti‐goat DyLight 649 (all from Jackson Immunoresearch, West Grove, PA).
Coronal or horizontal brain sections were prepared and processed for immunohistochemistry as described previously (Picardo et al.,
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