For histopathology, samples were formalin-fixed, paraffin-embedded, and sectioned at 5 μm thickness. For each sample, a section was stained using a standard hematoxylin and eosin (H&E) protocol. For IHC of cultured spheres, samples were sectioned at 10 μm thickness after fixation with 2% PFA in PBS for 2 hours on ice, and cryo-protected with 30% sucrose in PBS, followed by staining without heat antigen retrieval. Primary antibodies are listed in Table S2 . Fluorescently coupled secondary antibodies, including Alexa anti-rabbit 488, anti-mouse 488, and anti-rabbit 647 (Invitrogen, Grand Island, NY, USA) were used. Immunohistochemistry to detect cilia was performed on Myc, MycVD and Myc/ΔPOZ tumor sections and tumorspheres with antibodies to ARL13B and γ-tubulin (See Table S2 ) and with DAPI. Representative images of each sample/stain combination were captured and analyzed using Axiovision software (Carl Zeiss Microscopy, Thornwood, NY, USA). Immunoblotting was performed on tumor cells purified from mouse G3 MB (Myc), MycVD and Myc/ΔPOZ tumor, lysed in RIPA buffer and proteins separated on SDS/PAGE gels detected with primary antibodies against Myc, β-actin, and GAPDH (see Table S2 ), as previously described (Ayrault et al., 2010 (link); Kawauchi et al., 2012 (link)).
Anti rabbit 647
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-rabbit 647 is a fluorescent dye-labeled secondary antibody that specifically binds to rabbit primary antibodies. It can be used in various immunoassay techniques to detect and visualize target proteins or molecules recognized by rabbit antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse 488, by Thermo Fisher Scientific (4 mentions)
Anti rabbit 488, by Thermo Fisher Scientific (3 mentions)
Anti goat 647, by Thermo Fisher Scientific (3 mentions)
Rabbit anti dcp 1, by Cell Signaling Technology (2 mentions)
Mouse anti nubbin, by Developmental Studies Hybridoma Bank (2 mentions)
Mouse anti mmp1 c terminus, by Developmental Studies Hybridoma Bank (2 mentions)
Mouse anti lacz, by Developmental Studies Hybridoma Bank (2 mentions)
Anti rat 647, by Thermo Fisher Scientific (2 mentions)
Anti mouse 555, by Thermo Fisher Scientific (2 mentions)
Mouse anti ph3, by Cell Signaling Technology (2 mentions)
Chick anti gfp, by Abcam (2 mentions)
Axio imager m2, by Zeiss (2 mentions)
Mouse anti wg, by Developmental Studies Hybridoma Bank (2 mentions)
Ab70472, by Abcam (2 mentions)
Orca er ccd camera, by Hamamatsu Photonics (2 mentions)
Axioplan 2 microscope, by Hamamatsu Photonics (2 mentions)
Anti rabbit 568, by Thermo Fisher Scientific (2 mentions)
Anti guinea pig 555, by Thermo Fisher Scientific (2 mentions)
Airyscan lsm800, by Zeiss (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
13 protocols using anti rabbit 647
1
Histopathological and Immunohistochemical Analysis of Medulloblastoma
2
Characterizing Schwann Cell and Neurite Dynamics
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All samples were fixed at 4 days in vitro. Immunocytochemistry was completed to evaluate Schwann cell and neuronal phenotype, assess the presence of collagen, and characterize the cytoarchitecture within the micro-column and 2D cultures. Briefly, cultures were fixed in 4% formaldehyde for 30 min, washed in phosphate buffered saline (PBS), and permeabilized in 0.3% Triton X100 plus 4% normal horse serum (NHS) for 1 h. Cultures were incubated with primary antibodies in PBS + 4% serum solution) at 4°C for 12 h. To label Schwann cells, guinea pig anti-S100β (Synaptic Systems 287004; 1:500; intracellular calcium-binding protein) and rabbit anti-p-75 (Sigma N3908; 1:500; nerve growth factor receptor) were used. To evaluate neurite outgrowth, cultures were stained with mouse anti-beta tubulin III (Tuj1) (Sigma T8578; 1:500) to label all axons and neurons. To assess the distribution of collagen ECM, rabbit anti-collagen I (Abcam ab34710; 1:500) was used. After rinsing, appropriate secondary antibodies (1:500 in PBS + 4% NHS; anti-mouse 488, Invitrogen, A21202; anti-rabbit 488, Life Technology, A21206; anti-guinea pig 568, Sigma SAD4600038; and/or anti-rabbit 647, Invitrogen, A31573) were applied at room temperature for 2 h and Hoechst (Invitrogen H3570; 1:10,000) was then added to label all nuclei.
3
Immunofluorescent Staining of Larval Tissue
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Larvae were dissected in 1 x PBS followed by a 20-min fix in 4% paraformaldehyde in PBS. After 3 washes in 0.1% PBST (1 x PBS + 0.1%Triton-X), larvae were washed in 0.3% PBST and then blocked in 0.1% PBST with 5% normal goat serum (NGS) for 30 min. Primary staining was done overnight at 4°C, whereas secondary staining was done for 4 h at room temperature. The following primary antibodies were obtained from the Developmental Studies Hybridoma Bank: mouse anti-Nubbin (1:25), mouse anti-Wg (1:100), mouse anti-Mmp1 C-terminus (1:100), mouse anti-Mmp1 catalytic domain (1:100), mouse anti-LacZ (1:100), and rat anti-DE-cadherin (1:100). Rabbit anti-DCP1 (1:1000), and mouse anti-PH3 (1:400) were obtained from Cell Signaling Technologies, and chick anti-GFP (1:2000) was obtained from Abcam. The secondary anti-Chick 488 antibody was also obtained from Abcam. Other secondary antibodies, anti-rabbit 647, anti-rat 647, and anti-mouse 555 were obtained from Invitrogen. All secondary fluorophore-conjugated antibodies were used at 1:500. Images were obtained on a Zeiss AxioImager.M2 with ApoTome. For each experiment at least 15 discs were analyzed prior to choosing a representative image. Images were processed using Affinity Photo and Affinity Designer.
4
Immunostaining of Meiotic Chromosome Spreads
5
Immunostaining of Neuronal Subcellular Structures
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Antibody dilutions/amounts, validation, company names, catalogue numbers and clone numbers can be found either below, in the reporting summary, and/or supplementary table 1 . Cells were fixed for 20 min at RT in PBS with 4% formaldehyde freshly prepared from paraformaldehyde and blocked for 1 h in 50% SEA BLOCK and 0.5% Triton X‐100 solution. Neurons were incubated O/N at 4 °C in 20% SEA BLOCK and 0.5% Triton X‐100 in PBS with the following primary antibodies: GRP75 1:100 (Abcam, Cat #ab2799), KV2.1 10 µg/mL (NeuroMab, K89/34), CaV1.2 1:333 (Alomone, Cat #acc-003), KV2.1(pS603) 1:5 (L61/14.2), VDAC1 1:100 (Abcam, Cat #ab14734), SERCA 10 µg/mL (Abcam, Cat #ab2861), CaV1.3 10 µg/mL (Alomone Labs, Cat #ACC-005), pan-RyR 1:100 (Abcam, Cat #ab2868), IP3R 18 µg/mL (Abcam, Cat #ab5804), CaV2.1 1:200 (Alomone Labs, Cat #ACC-001), and KV2.1 pS603 1:5 (L61/14). After primary antibody incubation, neurons were washed 3 × 5 min and subsequently incubated for 1 h at RT with the following secondary antibodies: Goat anti-Mouse-647 and −568 nm 1:1000 (Invitrogen, Cat #A21236 and Cat #A11031, respectively), anti-Rabbit-647 and −555 nm 1:1000 (Invitrogen, Cat #A21245 and Cat #A21429, respectively), anti-Mouse IgG1-568 nm 1:1000 (Invitrogen, Cat #A21124) and anti-Mouse IgG1-CF568 1:250 (Sigma-Aldrich, Cat #SAB4600314).
6
Immunostaining of Meiotic Chromosome Spreads
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Meiotic cells were collected at indicated time points and fixation and chromosome spread was performed essentially as described using 4% paraformaldehyde42 (link). Immunostaining was performed as described42 (link). Primary antibodies were anti-PCNA (Abcam; ab70472, 1:100), anti-Zip3 antibody (a gift from Dr. Akira Shinohara, 1:400) and anti-Zip1 (a gift from Dr. Scott Keeney, 1:400); all incubated overnight at room temperature in 100 ul TBS/BSA buffer (10 mM Tris PH7.5, 150 mM NaCl, 1% BSA) Secondary antibodies were anti-rabbit 568 (A11036 Molecular Probes, 1:1000), anti-mouse 488 (A11029 Molecular Probes, 1:1000), anti-rabbit 647 (A21245 Invitrogen), and anti-guinea pig 555 (A21435 Life Technologies); all for 1 hr at 37˚C. Coverslips were mounted with Prolong Gold antifade reagent (Invitrogen, P36930). Digital images were captured using a Zeiss Airyscan LSM800 with Axiocam and analyzed using Zen (blue edition); or a Zeiss Axioplan II microscope, Hamamatsu ORCA-ER CCD camera and analyzed using Volocity software. Co-localization of protein foci was assigned to overlapping foci. Random colocalization (levels of colocalization by chance) were estimated by rotating the PCNA and Zip3 channels by 90° relative to one another and requantifying focus colocalization. Scatterplots were generated using the GraphPad program in Prism.
7
Immunohistochemical Analysis of Drosophila Larvae
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Larvae were dissected in PBS followed by a 20-minute fix in 4% paraformaldehyde in PBS. After 3 washes in 0.1% PBST (Triton-X), larvae were washed in 0.3% PBST and then blocked in 0.1% PBST with 5% NGS for 30 minutes. Primary staining was done overnight at 4 o C, while secondary staining was done for 4 hours at room temperature.
The following primary antibodies were obtained from the Developmental Studies Hybridoma Bank: mouse anti-Nubbin (1:25), mouse anti-Wg (1:100), mouse anti-Mmp1 C-terminus (1:100), mouse anti-Mmp1 catalytic domain (1:100), mouse anti-LacZ (1:100), and rat anti-DE-Cadherin (1:100). Rabbit anti-Dcp1 (1:1000) and mouse anti-PH3 (1:400) was obtained from Cell Signaling Technologies, and chick anti-GFP (1:2000) was obtained from Abcam. The secondary anti-Chick 488 antibody was also obtained from Abcam. Other secondary antibodies, anti-rabbit 647, anti-rat 647, and anti-mouse 555 were obtained from Invitrogen. All secondary fluorophore-conjugated antibodies were used at 1:500. Images were obtained on a Zeiss AxioImager.M2 with ApoTome. For each experiment at least 15 discs were analyzed prior to choosing a representative image.
Images were processed using Affinity Photo and Affinity Designer.
The following primary antibodies were obtained from the Developmental Studies Hybridoma Bank: mouse anti-Nubbin (1:25), mouse anti-Wg (1:100), mouse anti-Mmp1 C-terminus (1:100), mouse anti-Mmp1 catalytic domain (1:100), mouse anti-LacZ (1:100), and rat anti-DE-Cadherin (1:100). Rabbit anti-Dcp1 (1:1000) and mouse anti-PH3 (1:400) was obtained from Cell Signaling Technologies, and chick anti-GFP (1:2000) was obtained from Abcam. The secondary anti-Chick 488 antibody was also obtained from Abcam. Other secondary antibodies, anti-rabbit 647, anti-rat 647, and anti-mouse 555 were obtained from Invitrogen. All secondary fluorophore-conjugated antibodies were used at 1:500. Images were obtained on a Zeiss AxioImager.M2 with ApoTome. For each experiment at least 15 discs were analyzed prior to choosing a representative image.
Images were processed using Affinity Photo and Affinity Designer.
8
Whole Mount Immunohistochemistry of Ear Tissue
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Whole mount immunohistochemistry was performed on previously fixed tissue43 (link). Ears were washed in phosphate buffered saline (PBS), then washed five times five minutes in PBS/0.05% Tween20 followed by blocking for one hour in 5% normal donkey serum, 1% bovine serum albumin, and 0.5% TritonX-100 in PBS. The tissue was incubated in primary antibodies, diluted in blocking buffer, at 4°C for three nights. The following primary antibodies were used: MYO6 Rabbit (Sigma; 1:1000), MYOSIN7A Mouse (DSHB; 1:200), MYSOINVIIA Rabbit (Proteus Biosciences, Inc.; 1:500), Neurofilament 200 HC Chicken (Aves; 1:200), PROX1 Goat (R & D Systems; 1:200), and SOX2 Rabbit (Sigma; 1:500). Next, the tissue was washed four times thirty minutes, followed by overnight incubation at 4°C in secondary antibody in blocking buffer. Secondary antibodies were conjugated to Alexa flour anti-Mouse 488, anti-Rabbit 488, anti-Chicken 555, anti-Goat 647, or anti-Rabbit 647 (Life Tech; 1:1000). Nuclei were labeled using Hoescht Dye (1:2000), received as a gift from Bernd Fritzsch. Images were taken on either a Nikon C2 confocal microscope or a Leica Stellaris 5 confocal microscope and images were compiled in ImageJ and edited in CorelPhoto Paint (Version 19.0; 2017).
9
Flow Cytometric Characterization of Cells
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The cells were re-suspended in 2 mL FACS wash buffer and then filtered through a 40 μm mesh. Viable cells were estimated and the cells were then added to 1 mL of FACS wash and centrifuged for 3 minutes at 220 × g and 4°C. After removing the supernatant, the cells were incubated for 30 minutes on ice with either polyclonal rabbit anti-CD11b antibody (1:10) or polyclonal rabbit anti-Iba-1 (1:10) and polyclonal rabbit anti-NgR antibody (1:10), polyclonal rabbit anti-NgR2 and polyclonal rabbit anti-NgR3 antibodies (1:100) as primary antibodies, followed by 30 minute incubation on ice with anti-rabbit 647 (1:200), anti-rat 488 (Alexafluor 488 goat anti-rat IgG (1:200) and anti-goat 488 (1:200), Life Technologies) as secondary antibodies. The cells were also counterstained with DAPI (1:2000). Finally, the cell population was analyzed by using an FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Post-flow cytometric analysis was performed on FlowLogic (Inivai Technologies, Mentone, Australia).
10
Flow Cytometry Analysis of Immune Cells
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