For NK1-R immunostaining, renal cryostat sections (10 μm thick) on glass slides were fixed in acetone/methanol (1:1) and blocked with PBS containing 3% BSA and 10% rabbit normal serum. Subsequently, slides were incubated with a primary antibody raised against the amino terminus region of human NK-1R (Santa Cruz Biotechnologies, Heidelberg, Germany) and specific markers for macrophages (mouse anti-rat ED1; Serotec, Düsseldorf, Germany) as well as DCs (mouse-anti-rat CD11c antibody; Serotec, Düsseldorf, Germany). Eventually, binding sites of NK-1R were detected using Cy3-conjugated rabbit-anti-goat antibody (1:500; Jackson ImmunoResearch/Dianova). Detection of ED1 and CD11c was performed using an Alexa488-conjugated donkey-anti-mouse antibody (Molecular Probes, Invitrogen, Karlsruhe, Germany).
Cy3 conjugated goat anti rabbit antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3-conjugated goat anti-rabbit antibody is a secondary antibody conjugated with the fluorescent dye Cy3. It is designed to bind to and detect rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Mowiol, by Merck Group (2 mentions)
Cellp software, by Olympus (2 mentions)
Alexa 488 conjugated donkey anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti rat ed1, by Bio-Rad (1 mentions)
Ab18058, by Abcam (1 mentions)
Donkey anti goat antibody, by Santa Cruz Biotechnology (1 mentions)
Af2199, by R&D Systems (1 mentions)
Nb100 134, by Novus Biologicals (1 mentions)
Anti mouse, by LI COR (1 mentions)
Af2886, by R&D Systems (1 mentions)
A freezing microtome, by Leica camera (1 mentions)
Anti ki67 antibody, by Abcam (1 mentions)
Anti ve cadherin antibody, by Abcam (1 mentions)
Anti vwf antibody, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit antibody, by Jackson ImmunoResearch (1 mentions)
Leica fluorescence microscope, by Leica camera (1 mentions)
Anti cd31 antibody, by Abcam (1 mentions)
Anti ddx3, by Santa Cruz Biotechnology (1 mentions)
Anti eif4e, by Santa Cruz Biotechnology (1 mentions)
Irdye 800 conjugated goat anti mouse igg, by LI COR (1 mentions)
18 protocols using cy3 conjugated goat anti rabbit antibody
1
NK1-R Immunostaining in Renal Cryostat Sections
2
Antibody profiling for Western blot and IF
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Goat polyclonal antibody against human legumain (LGMN) was used for Western blotting, and immunofluorescence was purchased from R&D Systems Inc. (Bio-Techne GmbH, Wiesbaden, Germany, #AF2199). Rabbit monoclonal antibodies against hexokinase 2 (HK2, #2867), lactate dehydrogenase (LDHA, #3582), β-tubulin (#2128) and β-Actin (#4970) were purchased from Cell Signaling Technology Europe B.V. (Frankfurt, Germany). A mouse monoclonal antibody against Vinculin (#ab18058) was purchased from Abcam (Cambridge, UK). For HIF detection, a polyclonal rabbit antibody against HIF-1α (#NB100-134) from Novus biologicals (Bio-Techne GmbH, Wiesbaden, Germany) and a polyclonal goat antibody for HIF-2α (#AF2886) from R&D systems Inc. were used. A rabbit antibody against nucleolin was obtained from Santa Cruz Biotechnology (Heidelberg, Germany, #sc-13057). The primary antibody used for immunofluorescence was detected with a Cy3-conjugated rabbit anti-goat antibody (Jackson ImmunoResearch, Ely, UK). The primary antibodies used for Western blotting were detected with an HRP-conjugated goat anti-rabbit (Dako, Agilent Technologies, Waldbronn, Germany), donkey anti-goat antibody (Santa Cruz Biotechnology, Heidelberg, Germany) or anti-mouse (LI-COR Biosciences, Bad Homburg, Germany) antibody.
3
Immunohistochemistry of Microvascular Markers
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After fixation overnight, eyes were cut into 15 µm coronal sections using a
freezing microtome (Leica, Solms, Germany). First, the sections were incubated
in blocking buffer containing 10% normal goat serum and 0.2% Triton X-100
dissolved in phosphate-buffered serum (PBS) for 1 h at room temperature. With
the primary antibodies, the sections were probed by anti-CD31 antibody (1:50
dilution, Abcam, Cambridge, UK), anti-vWF antibody (1:300 dilution, Abcam),
anti-VE-cadherin antibody (1:500 dilution, Abcam) or anti-Ki-67 antibody (1:100
dilution, Abcam) overnight at 4°C following by incubation with Alexa Fluor
488–conjugated goat anti-rabbit antibody (1:500 dilution, Jackson ImmunoResearch
laboratories, Inc, West Grove, USA) or Cy3-conjugated goat anti-rabbit antibody
(1:500, Jackson ImmunoResearch laboratories, Inc) for 1 h at room temperature.
Then the sections were mounted on glass slides and visualized with Leica
fluorescence microscope (DMI3000, Leica). A slide without primary antibody was
regarded as the negative control. Exposure conditions in the same channel of
different groups in each experiment were kept the same.
freezing microtome (Leica, Solms, Germany). First, the sections were incubated
in blocking buffer containing 10% normal goat serum and 0.2% Triton X-100
dissolved in phosphate-buffered serum (PBS) for 1 h at room temperature. With
the primary antibodies, the sections were probed by anti-CD31 antibody (1:50
dilution, Abcam, Cambridge, UK), anti-vWF antibody (1:300 dilution, Abcam),
anti-VE-cadherin antibody (1:500 dilution, Abcam) or anti-Ki-67 antibody (1:100
dilution, Abcam) overnight at 4°C following by incubation with Alexa Fluor
488–conjugated goat anti-rabbit antibody (1:500 dilution, Jackson ImmunoResearch
laboratories, Inc, West Grove, USA) or Cy3-conjugated goat anti-rabbit antibody
(1:500, Jackson ImmunoResearch laboratories, Inc) for 1 h at room temperature.
Then the sections were mounted on glass slides and visualized with Leica
fluorescence microscope (DMI3000, Leica). A slide without primary antibody was
regarded as the negative control. Exposure conditions in the same channel of
different groups in each experiment were kept the same.
4
Antibody Characterization in BAdV-3 Infected Cells
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Anti-pVIIIa recognizes a protein of 24 kDa in BAdV-3 infected cells (Ayalew et al., 2014 (link)). Anti-DBP recognizes a protein of 48 kDa in BAdV-3 infected cells (Zhou et al., 2001 (link)). Anti-DDX3, anti-eIF4G, anti-eIF4E, anti-eIF3, anti-PABP and fluorescence conjugated goat anti-mouse IgG-FITC (Santa Cruz Biotechnology, Inc., USA), Cy-3 conjugated goat anti-rabbit antibody (Jackson Immuno Research), Alexa Flour 680 goat anti-rabbit IgG antibody (Molecular Probes) or IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) and anti-HA and anti-β-actin MAb (Sigma Aldrich) were purchased.
5
Cre-Mediated Expression of 5-HT Receptors
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To express HA-tagged 5-HT2B and Flag-tagged 5-HT1A specifically in 5-HT neurons, we use a Double floxed Inverse Orientation (DIO) adeno-associated virus (AAV) construct that allows Cre-mediated expression of the transgene (pAAV-EF1A-DIO-WPRE-pA vector; RRID Addgene_39320)24 (link) (Figure S2 ). The viruses packaged into AAV2.9 serotype with titers of 1012-1013 viral particles/mL were obtained (UNC Vector Core, Chapel Hill USA). Both AAVs were stereotaxically injected in the B7 raphe nuclei of males or females Pet1-Cre+/0 mice at 6 weeks of age.50 (link) They were used 3 weeks after surgery for histological procedures. Respective expression was assessed by immunostaining using a mouse anti-HA antibody (1:500; Cell Signaling#2367, RRID AB_10691311 and a rabbit anti-Flag (1:500; Cell Signaling#14793 RRID AB_2572291 followed by Cy5-conjugated donkey anti-mouse (1.9 μg/mL; Jackson ImmunoResearch#715-175-150, RRID AB_2340819 ) and the Cy3-conjugated goat anti-rabbit antibody (1.9 μg/mL; Jackson ImmunoResearch#111-165-003, RRID AB_2338000 ).
6
Immunostaining of Agrin in Cells and Tissues
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Immunocytochemistry and immunohistochemistry were performed as previously described.25 (link) For antigen retrieval on paraffin sections we used pepsin digestion.25 (link) The primary anti-agrin rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, Texas, USA) or the isotype-matched negative control normal rabbit IgG (Dako, Ely, Cambridgeshire, UK) was diluted 1:200 in blocking buffer. Secondary antibodies were cy3-conjugated goat anti–rabbit antibody diluted 1:300 (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA) for immunocytochemistry or goat anti-rabbit antibody Alexa 555 diluted 1:300 (Life Technologies) for immunohistochemistry. Slides were mounted in Mowiol (EMD Millipore, Darmstadt, Germany), and images were acquired with a fluorescence microscope (BX61; Olympus, Southend-on-Sea, UK) using a Uplan-Fluor 40× NA 0.85 objective lens. Images were acquired by using an F-View II Soft Imaging Solutions (SIS) camera and Cell P software (Olympus). After acquisition, the contrast of the images was enhanced for best graphic rendering using Photoshop 7.0, all with the same parameters, without altering the relationship of the target to control the images.
7
Immunofluorescence Analysis of Primary Hepatocytes
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Primary hepatocytes were fixed in 4% paraformaldehyde and made into smear. Immunofluorescence was performed as previous described [9 (link)]. Albumin antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD31 antibody (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Laminin antibody (1:100, Abcam, Cambridge, UK), and Nestin antibody (1:50, Chemicon, Billerica, MA, USA) were used. FITC-conjugated donkey anti-goat antibody or Cy3-conjugated goat anti-rabbit antibody was used as secondary antibodies (1:100, Jackson Immuno-Research, West Grove, PA). At last, nuclei were stained with DAPI.
8
Immunostaining of Zebrafish Embryos
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Zebrafish embryos at the appropriate developmental stage were fixed overnight in 2% paraformaldehyde (PFA) in PBS at 4°C. After washing with 1 × PBS–Triton X-100 (0.1%; PBS-T) and blocking in 10% goat serum in 1 × PBST (blocking buffer;BB), embryos were incubated overnight at 4°C with rabbit anti-DsRed (1:500 in BB; Takara Bio 632496), mouse anti-Myh6 antibody (1:200 in BB, DSHB, S46), or chicken anti-GFP (1:500 in BB, Aves Labs, GFP-1010). After washing in PBST, the embryos were incubated overnight at 4°C in Cy3-conjugated goat anti-rabbit antibody (1:500 in BB; Jackson Immunoresearch, 111-165-144), Alexa488-conjugated goat anti-mouse (1:500 in BB, Invitrogen, A21133) or Alexa488-conjugated goat-anti-chicken (1:500 in BB; Invitrogen, A11039). Embryos were washed in PBST before imaging.
9
Immunofluorescence analysis of Cx40 in transfected HeLa cells
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HeLa cells were grown on glass coverslips and transiently transfected with pIRES2-EGFP Cx40 WT and mutant constructs. 24 h after transfection, cells were fixed with 1% paraformaldehyde in PBS for 15 min, permeabilized with PBS-0.1%Triton X-100 for 10 min, and then blocked with 3% BSA in PBS-0.1% Triton X-100 for 30 min at room temperature. A 1:100 dilution of a polyclonal rabbit anti-Cx40 antibody (Santa Cruz Biotechnology, Inc.) was applied for 1 h. Then, cells were treated with a 1:2,000 dilution of secondary Cy3-conjugated goat anti–rabbit antibody (AffiniPure; Jackson ImmunoResearch Laboratories, Inc.) for 30 min in the dark. The coverslips were mounted on slides using Vectashield with DAPI (Vector Laboratories) and visualized with 40 or 60× objectives on a microscope (BX51; Olympus) and photographed with a digital camera (MagnaFire; Olympus).
10
Immunohistochemical Staining of FMRFamide
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