Frozen spinal sections were processed for immunohistochemistry followed method described in our recent articles [35 (link), 39 (link)]. The tissue sections were incubated with primary antibodies, followed by respective 2nd antibodies for histological evaluation as described. Primary antibodies used were: GFP (1:200, Millipore, Billerica, MA), iba1(1:400, Abcam, Cambridge, UK), ED1 (1:250, AbD Serotec, UK), GFAP (1:400; DakoCytomation,Glostrup,Denmark), S100 beta (1:250; Sigma), p75NGFR(+) (1:500; Millipore, Billerica, MA) and type III beta tubulin (1:250; Covance Research Products Inc., Denver, PA). For double immunostaining, the secondary antibodies used were Alex 488 fluorophore donkey anti-rabbit antibody (1:200; Molecular Probes, Eugene, OR, USA) and Cy3-conjugated donkey anti-mouse antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA). Primary antibody omission controls were performed for all immunostaining protocols to control for nonspecific binding. Fluorescent visualization and photography were performed on a Zeiss Axioscope microscope with appropriate filter sets (Zeiss, Oberkochen, Germany).
Cy3 conjugated donkey anti mouse antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3-conjugated donkey anti-mouse antibody is a secondary antibody that binds to mouse primary antibodies. The Cy3 fluorescent dye is conjugated to the antibody, allowing for the detection and visualization of mouse target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Iba 1, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Ab26116, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Santa Cruz Biotechnology (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
E800 microscope, by Nikon (1 mentions)
Alexa 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
β catenin, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dylight 405, by Jackson ImmunoResearch (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
C2 laser scanning confocal microscope, by Nikon (1 mentions)
Sp2 confocal microscope, by Leica (1 mentions)
Hoechst, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
Automatic slide scanner 250 flash, by 3DHISTECH (1 mentions)
Bond max system, by Leica (1 mentions)
9 protocols using cy3 conjugated donkey anti mouse antibody
1
Immunohistochemical Analysis of Spinal Tissue
2
Immunostaining and Western Blot Analysis
3
Immunofluorescent Analysis of Active β-Catenin
4
Quantifying PVBC and CCK/CB1BC Distribution
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To estimate the target distribution of PVBCs using light microscopy, sections were incubated in mouse anti-Kv2.1, which was visualized with Cy3-conjugated donkey anti-mouse antibody (1:200, Jackson ImmunoResearch Laboratories Inc.). In the case of CCK/CB1-expressing BCs (CCK/CB1BCs), sections were incubated in a mixture of mouse anti-Kv2.1 and rabbit anti-CB1 primary antibodies visualized with Alexa 488 (donkey anti-mouse, 1:200, Molecular Probes) and DyLight 405 (donkey anti-rabbit, 1:200, Jackson ImmunoResearch Laboratories Inc.). Following several washes in TBS, sections were mounted on glass slides in Vectashield (Vector Laboratories). Images were taken using an A1R or a C2 confocal laser scanning microscope (Nikon Europe, Amsterdam, The Netherlands) using a 60 × (NA = 1.4) apochromatic objective (z step size: 0.13 μm, xy: 0.06 μm/pixel).
5
Cocaine Treatment Effects on Receptor Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK-293T cells were treated with 30 μM cocaine or vehicle for 30 min, then were washed with PBS, fixed in 4% paraformaldehyde for 15 min and washed with PBS containing 20 mM glycine to quench free aldehyde groups. After permeabilization with PBS-glycine buffer containing 0.2% Triton X-100 for 5 min, cells were blocked with PBS containing 1% bovine serum albumin (BSA) for 1 h at room temperature. D1R-YFP and D2R-YFP were detected by its own fluorescence (wavelength 530 nm), and σ2R-Rluc was stained using a primary anti-Rluc mouse monoclonal antibody (1/200, Millipore, CA, United States) for 1 h, washed and stained for another hour with the secondary Cy3-conjugated donkey anti-mouse antibody (1/200, Jackson Immunoresearch Laboratories, West Grove, PA, United States). Nuclei were stained with Hoechst (1/100, Sigma–Aldrich, St. Louis, MO, United States) and then samples were rinsed several times and mounted with Mowiol 30% (Calbiochem). Images were taken using a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany).
6
Immunofluorescence Staining of Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H9c2 CMs were fixed with 2% paraformaldehyde at RT for 30 min and then permeated with 0.1% TX-100 at RT for 15 min. After being blocked with 1% BSA and 2% donkey serum for 1 h, the cells were incubated with β-MHC antibody (1∶50; Millipore, MA) overnight at 4°C, and followed by incubation with Cy3-conjugated donkey anti-mouse antibody (1∶250; Jackson, PA) at RT in the dark for 1 h. DAPI was used for nuclear stain. Images were obtained with an inverted microscope.
7
Immunocytochemical Analysis of HIF-1α and DKK-1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunocytochemistry was performed as previously described (22 (link)). Briefly, the synovial fibroblasts 1×106/well were plated in six-well culture plates that contained carry sheet glass. Once the cells on the glass slide had reached 70% confluence, they were incubated with a mixture of the IL-1β or rhDKK-1 and serum-deprived DMEM. Following 24 h incubation, the cells were fixed for 10 min with 4% formaldehyde in phosphate-buffered saline (PBS; pH 8.0). Following three washes with PBS, the cells were permeabilized for 5 min with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS, prior to being thoroughly washed again with PBS. PBS supplemented with 10% FBS was used to block non-specific binding for 60 min. The cells were subsequently incubated with HIF-1α (1:100) and DKK-1 antibodies (1:50) overnight at 4°C. The cells were washed thoroughly three times with PBS, and then incubated with Cy3-conjugated donkey anti-mouse antibody (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at room temperature for 60 min, followed by further washing three times with PBS. The nuclei were then stained with DAPI, and images of the cells were captured and analyzed by CLSM-310 confocal laser scanning microscopy (Carl Zeiss Inc., Thornwood, NY, USA).
8
Spinal Cord and Ganglia Histology in Rats
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fourteen to 28 days after surgery, rats were terminally anesthetized with thiopental and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The lumbar spinal cord and lumbar spinal ganglia (L3–L5) were removed, post-fixed for 2 h, and cryoprotected overnight in phosphate-buffered saline (PBS) with 30% sucrose added. Then, 20-μm-thick transverse sections of the lumbar spinal cord were cut using a freezing microtome and processed for histochemistry. Free-floating sections were washed in PBS and incubated overnight with combinations of mouse monoclonal anti-CD11b (OX42) antibody (1:1000, Abd Serotec/Bio-Rad, Hercules, California, USA) and biotin-conjugated Bandeiraea simplicifolia isolectin B4 (IB4) lectin (1:500, Sigma-Aldrich, Saint Louis, USA) in PBS with 1% Triton X100 (TPBS). Sections were rinsed in PBS (3 × 5 min) and incubated in a mixture of Cy3-conjugated donkey anti-mouse antibody (1:500, Jackson ImmunoResearch, West Grove, PA, USA) and FITC-conjugated ExtrAvidin (1:500, Jackson ImmunoResearch, West Grove, PA, USA) in TPBS. Sections were incubated for 2 hours at room temperature, rinsed in PBS (3 × 5 min), mounted on slides, and covered with Prolong Gold mounting medium (Invitrogen, Carlsbad, California, USA).
9
Immunofluorescence and Immunohistochemistry of Lung Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microtubule-associated protein 1 light chain 3 (LC3) detecting antibody (rabbit polyclonal, Novus) and Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch) were used. Images were collected using LSM 700 laser scanning confocal system, attached to an upright motorized microscope with × 63/1.40 oil objective (ZeissAxio Imager Z2). For detection of T-cells, frozen sections (7 µm thick) were stained with anti-CD8 primary antibody (YTS169.4, abcam), followed by secondary Cy3-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch). The whole slide image was collected using automatic slide scanner 250 Flash (3DHISTECH). For immunohistochemistry staining, lungs were embedded in paraffin and staining was performed on 4-µm sections using the Leica Bond max system (Leica Biosystems Newcastle Ltd, UK). Sections were dewaxed and pretreated with epitope retrieval solution (Leica Biosystems Newcastle Ltd, UK) followed by anti-CD45 antibody (1:600, ab10558 by Abcam).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!