Drosophila S2 cells were plated on ibidi dishes (Planegg/Martinsried, Germany) or coverslips were fixed with 3% paraformaldehyde solution (pH 7.3) at 21°C for 10 min. In some cases, fixed cells were subsequently permeabilised by treatment with 0.1% Triton X-100 at 21°C for 10 min. In cases where antigen retrieval was required, proteins were denatured by the treatment of fixed and permeabilised cells with 6 M guanidinium hydrochloride (GdnHCl) at 21°C for 7 min. Cells were subsequently rinsed with either PBS in the case of non-detergent-treated cells or in the case of detergent-treated samples, with 0.01% Triton X-100 in PBS (PBST). Washed cells were incubated with mouse monoclonal anti-PrP antibody 4H11 [55 (link)] and the Golgi-specific rabbit polyclonal antibody GM130 (Abcam, Cambridge, U.K.) at 37°C for 60 min. After three washing steps in PBS or PBST, as appropriate, cells were incubated with Alexa Fluor-488-conjugated goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany) or Cy3-conjugated goat anti-rabbit IgG (Dianova, Hamburg, Germany) at 21°C for 60 min. Nuclei were stained with Hoechst DNA staining dye (Sigma, Taufkirchen, Germany). Confocal laser scanning microscopy was performed on an LSM 700 laser scanning microscope (Zeiss, Göttingen, Germany).
Cy3 conjugated goat anti rabbit igg
Manufactured by Dianova
Sourced in Germany
Cy3-conjugated goat anti-rabbit IgG is a secondary antibody that binds to rabbit immunoglobulin G (IgG) and is conjugated to the Cy3 fluorescent dye. This product can be used for detection and visualization of rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 700 laser scanning microscope, by Zeiss (1 mentions)
Hoechst dna staining dye, by Merck Group (1 mentions)
Gm130, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
On targetplus non targeting control pool, by Horizon Discovery (1 mentions)
Rabbit polyclonal igg, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Carl Roth (1 mentions)
Zen software, by Zeiss (1 mentions)
Anti e coli lps antibody, by Abcam (1 mentions)
Anti tlr4, by Thermo Fisher Scientific (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Bx61wi fluoview 1000 fv1000, by Olympus (1 mentions)
5 protocols using cy3 conjugated goat anti rabbit igg
1
Immunofluorescence Staining of Drosophila S2 Cells
2
Dynamin Knockdown Impacts TNR Epitope Labeling
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The neurons were co-transfected with 50 nM dynamin 1, 2, and 3 siRNA constructs (previously described in65 (link)) or with non-targeting control siRNA (ON-TARGETplus Non-targeting Control Pool; # D-001810-10, Horizon Discovery) at DIV 7, using Lipofectamine RNAiMAX transfection reagent (#13778030; Thermo Fisher Scientific, USA). At DIV 14, labeling of newly-emerged TNR epitopes was performed as in the other experiments (see ‘Blocking-labeling assay and live treatments’). Fixation and immunostaining were performed as in other experiments (see ‘Fixation and post-fixation immunostaining’), using 1:100 rabbit anti-dynamin 1, 2, 3 (#115 002, Synaptic Systems, Göttingen, Germany), followed by 1:200 Cy3-conjugated goat anti-rabbit IgG (#111-165-144; Dianova, Germany).
3
Immunofluorescent Detection of POR Protein
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Cells growing on glass slides were fixed for 30 min in 5% formaldehyde solution, followed by 10 min permeabilisation in 0.2% Triton-X100 solution. Blocking of unspecific binding sites occurred for 30 min in 3% BSA solved in PBS. All steps were performed at ambient temperature. Visual detection of POR protein expression was realised using polyclonal rabbit-anti-POR IgG as primary antibody (1 mg/mL, Abcam) at 1:100 dilution and a Cy3 conjugated goat-anti-rabbit IgG (1:200, Dianova GmbH, Hamburg, Germany) as secondary antibody. Incubation of the samples with the primary antibody occurred over night at 4°C and with the secondary antibody for 1 hour at ambient temperature. For assessment of background binding, samples were stained with a primary antibody of the same isotype (polyclonal rabbit IgG, 1 mg/mL, Abcam) but without target binding properties serving as isotype control. Staining of genomic DNA was realised by 5 min incubation of the samples with 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, 0.2 μg/mL in PBS, Carl Roth GmbH + Co. KG, Karlsruhe, Germany). High resolution pictures were taken by using a LSM800 confocal Laser Scanning Microscope system and ZEN software for picture post processing (Carl Zeiss Microscopy GmbH, Jena, Germany).
4
Antibodies Production for E. coli Research
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Anti-E. coli O104 LPS and H4 antibodies were produced50 in rabbits using reference strains H519 (O104:K−:H12) and U9-41 (O2:K1:H4), respectively. Anti-Stx2a24 (link) and anti-OmpA51 (link) antibodies were described. Rabbit antibodies against SigA, Pic, SepA, and ShET1 A subunit were produced by Aptum Biologics Ltd. (Southampton, UK), and antibody against AAF/I A subunit by Davids Biotechnologie (Regensburg, Germany). Commercial antibodies were: anti-actin (Santa Cruz Biotechnology); anti-E. coli LPS antibody (Abcam); anti-TLR5 neutralising antibody and the rat IgG control (InvivoGen); anti-TLR5 detection antibody (Abcam); anti-TLR4 (Invitrogen); anti-MD-2 (Novus Biological); Alexa Fluor 488-conjugated goat anti-rabbit or anti-mouse IgG (Molecular Probes); Cy3-conjugated goat anti-rabbit IgG; and alkaline-phosphatase-conjugated goat anti-rabbit IgG (Dianova).
5
Microglia density and neuronal envelopment analysis
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After fixation by perfusion, as above, the hippocampi were dissected, postfixed in 4% PFA and subsequently cryoprotected. The hippocampi were cryo‐sectioned at 30 μm and after a blocking and permeabilization step the sections were incubated overnight with anti‐Iba1 primary antibodies (dilution of 1:1,000; Synaptic Systems, #234003), then with a Cy3‐conjugated goat anti‐rabbit IgG (1:500, Dianova) and mounted using an anti‐fading mounting medium. For analyzing changes in microglia density z‐stacks of 3 to 5 regions of interest (ROIs) per mouse were imaged using a 20x objective (NA 0.8) at a z‐step of 1 μm. The number of microglia was counted using the multipoint tool of ImageJ and is expressed as number of cells per mm3 of tissue. For analyzing the enwrapping of microglia around neuronal cell bodies, confocal images of at least 10 randomly selected eGFP‐expressing CA1 pyramidal neurons were analyzed with a BX61WI FluoView 1000 (FV1000) Olympus confocal microscope. Stacks were acquired using a 40x oil objective (NA 1.3), z‐steps of 1 μm. The neuron profile as well as the length of Iba1 positive microglia processes contacting the neuronal cell body were measured using the freehand tool from ImageJ for each z‐plane and summed to obtain one value of overlap per each neuron (expressed in % of the neuronal perimeter).
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