Sperm cells from the cauda epididymis attached to glass coverslips were fixed in 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 10 mins, washed with PBS and blocked with 10% goat serum for 1h. Samples were stained overnight with indicated primary antibody in 10% goat serum at 4°C. For secondary antibodies, Alexa568 or Alexa488 conjugated goat-anti-rabbit (Invitrogen) were used. Images were acquired by confocal microscopy (Zeiss LSM710 Elyra P1).
Alexa 488 conjugated goat anti rabbit
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor® 488-conjugated goat anti-rabbit is a secondary antibody that specifically binds to rabbit primary antibodies. Alexa Fluor® 488 is a fluorescent dye that can be detected using standard green fluorescence detection methods.
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Lab products found in correlation
Cy3 conjugated goat anti mouse, by Jackson ImmunoResearch (5 mentions)
Alexa568 conjugated goat antimouse, by Thermo Fisher Scientific (4 mentions)
Alexa 546 conjugated goat anti mouse, by Thermo Fisher Scientific (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Alexa 568, by Thermo Fisher Scientific (2 mentions)
Lsm710 elyra p1, by Zeiss (2 mentions)
Tcs sp5 x confocal microscope, by Leica (2 mentions)
A11122, by Merck Group (2 mentions)
Gold anti fade mounting solution, by Thermo Fisher Scientific (2 mentions)
Rabbit monoclonal anti rad51 antibody, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 510 confocal microscope, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Tcs sp5, by Leica (2 mentions)
Alexa488 conjugated goat anti mouse, by Jackson ImmunoResearch (2 mentions)
40 protocols using alexa 488 conjugated goat anti rabbit
1
Immunofluorescence Staining of Sperm Cells
2
ICC Cell Culture and Immunocytochemistry
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For ICC, the cells (5 × 104 cells/mL) were cultured on poly-L-lysine-coated aclar plastic coverslips as described previously [29 (link)]. Medium containing 1% FBS (EMD Millipore Corp.) was changed to medium containing the HDAC inhibitors. The cells were fixed with 4% paraformaldehyde (PFA; T&I Co., Chuncheon-si, Republic of Korea) for 15 min at room temperature. The cells were then blocked with 0.5% Triton X-100 (Sigma–Aldrich) for 20 min and 10% normal goat serum (NGS; Vector Laboratories, Inc., Burlingame, USA) for 30 min. Primary antibodies were added for 1.5 h, and the secondary antibodies were then added and kept in the dark for 1 h. For staining nuclei, 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL, Life Technologies Corporation) was added for 30 min. The cells were then imaged with a Zeiss LSM510 confocal microscope (Carl Zeiss, Jena, Germany) after mounting [29 (link)]. The primary antibodies used were microtubule-associated protein 2 (MAP2, 1:200; Cell Signaling Technology, Danvers, USA) and neurofilament-H (NFH, 1:400; Cell Signaling Technology). Alexa 488-conjugated goat anti-rabbit (Invitrogen Co., Waltham, USA) and Alexa 594-conjugated goat anti-mouse (Invitrogen Co.) were used as the secondary antibodies. All experiments were repeated at least three times.
3
Quantitative Apoptosis and Proliferation Assay
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The ApopTag Red In Situ Apoptosis Detection Kit (Merck Millipore, S7165) was used to detect apoptotic cells. For cell proliferation, anti-phospho-histone H3 (H3S28P; 1:250, Merck Millipore) followed by Alexa 488-conjugated goat anti-rabbit (1:200, Invitrogen) was used. Nuclear staining was achieved by a 20-min incubation in Hoechst 33342 (Invitrogen). Images were collected by confocal microscopy (8 μm/section; LSM710 ConfoCor 3, Carl Zeiss) and processed using ImageJ software. Total numbers of TUNEL- and H3S28P-positive profiles were counted in 7 consecutive sections. TUNEL- and H3S28P-positive profiles were automatically detected using the following set of parameters in Volocity Image Analysis Software (Perkin-Elmer): (1) Find Objects by Intensity; (2) exclude objects smaller than 7 μm2; (3) separate touching objects greater than 25 μm2. The apoptotic index (TUNEL) and mitotic index (H3S28P) were calculated as the percentage of cells positive for each marker to the total number of Hoechst 33342 -positive (nuclei marker, blue) cells in ectoderm (epiblast and extraembryonic ectoderm) and embryonic visceral endoderm per embryo. All graphs were generated using GraphPad Prism version 7 and data are shown as mean and SEM. Mann-Whitney U test was used for analysing cell number; TUNEL and H3S28P profiles and ***p < 0.001 was considered highly significant.
4
Immunofluorescent Imaging of Murine Hippocampus
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The procedures were modified from the previous reports (Chen et al., 2021 (link)). Mice were anesthetized with 1% pentobarbital sodium and intracardially perfused with saline followed by 4% polyformaldehyde. The brains were removed, postfixed in 4% polyformaldehyde for 12 h, and then equilibrated in 30% sucrose. Coronal sections of 40-μm thickness containing the hippocampus were collected. Brain slices were blocked for 1 h with 5% bovine serum albumin and 1% Triton X-100 at room temperature and then incubated with primary antibodies (rabbit anti-NeuN 1:300, Cell Signaling Technology, Cat# 12943; mouse anti-GFAP 1:500, Cell Signaling Technology, Cat# 3670 and rabbit anti-Iba1 1:500, Cat# 01919741) overnight at 4°C. The next day, the slices were incubated with secondary antibodies (Alexa 488-conjugated goat anti-rabbit and Alexa 647-conjugated goat anti-mouse, 1:500, Invitrogen) for 2 h at room temperature after washing three times with PBS and then incubated with DAPI (Invitrogen) for 10 min and washed for another three times. The slices were mounted on microscope slides. Fluorescence images were captured using a confocal microscope (A1R, Nikon). Cell counting and fluorescent area calculation were performed by ImageJ.
5
Immunostaining of Drosophila Brain Tissues
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Brains were dissected in PBS and fixed in PBS with 4% paraformaldehyde at room temperature for 20 min. They were washed three times, 20 min each in PBS containing 0.5% Triton-X100 (PBT), followed by 30 min incubation in PBT containing 5% normal goat serum. Anti-GFP (1:1000; Invitrogen, A11122), anti-TH (1:200; Millipore, AB152) and anti-nc8239 (link)(1:50; DSHB) antibodies were added to the solution and brains were incubated overnight at 4°C. Brains were then washed in PBT three times, 20 min each at room temperature, followed by incubation in PBT containing Alexa 488 conjugated goat anti-rabbit (1:100; Invitrogen, A11034) and Cy3 conjugated goat anti-mouse (1:200; Jackson ImmunoResearch, 115-165-003) overnight at 4°C. Brains were then washed in PBT three times, 20 min each at room temperature, before being mounted on slides with Gold anti-Fade mounting solution (Invitrogen). Imaging was performed using a Leica TCS SP5X confocal microscope. The resolution of the image stack was 1024 × 1024 with 1μm step size. Images were processed using Fiji. For each genotype, at least two brains were dissected to confirm that they had the same pattern of expression.
6
Immunostaining of Drosophila Brain Tissues
7
Quantifying DNA Damage and Repair Foci
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Cells were treated for 24 h with control vehicle (DMSO) and 1 μM VE-821 with or without 10 μM olaparib. Cells were stained with mouse monoclonal anti phospho-Histone H2A.X (Ser139) antibody (SCBT) at 1:500 and rabbit monoclonal anti RAD51 antibody (CST: 8875S) at 1:250. Secondary antibodies used were Alexa 488 conjugated goat anti rabbit and Alexa 546 conjugated goat anti mouse (Invitrogen, Thermo Fisher Scientific), both at 1:1000. Cells were imaged using a Leica DM6 microscope and Leica Application Suite (LAS) X software (Leica Microsystems, Wetzlar, Germany). The number of RAD51 foci in each cell and total nuclear fluorescence intensity for γH2AX were quantified using ImageJ software (Version 1.52p; Java 1.8.0_172 (64-bit)) and data was plotted using GraphPad Prism v6 (San Diego, CA, USA).
8
Immunofluorescence Microscopy of RNLS and αSMA
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The slides were deparaffinized and processed as above for immunofluorescence microscopy. After antigen retrieval, sections were blocked with TBS/0.3% TritonX-100/10% goat serum) for 1 hr and incubated with a cocktail of m28-RNLS (1:100) plus α-smooth-muscle actin (αSMA) mouse monoclonal (1:400 Sigma-Aldrich, St. Louis, MI, USA) at 4°C overnight. Alexa 488-conjugated goat anti-rabbit (1:2000, Invitrogen Corporation, Carlsbad, CA) and Alexa 594-conjugated goat anti-mouse (1:2000, Invitrogen Corporation, Carlsbad, CA) were used to detect the primary antibodies. Tissue autofluorescence was quenched using a commercial preparation (Vector TrueVIEW Autofluorescence Quenching Kit, Vector Laboratories, Burlingame, CA, USA). The specificity of labeling on each tissue was confirmed by labeling tissue sections in the absence of the m28-RNLS and αSMA primary antibodies.
9
Immunofluorescent Labeling of Spiral Ganglion Neurons
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Slips of SGNs were fixed in 4% paraformaldehyde 30 min and permeabilized in 0.1% TrionX-100 15 min. After washing by PBS, they were incubated in blocking solution of bovine serum albumin (BSA, 5%, Sigma, USA) 20 min and in antibodies against AIF (1:200, rabbit, Abcam, USA) and β-tubulin (1:200, mouse, Abcam, USA) 24 h overnight at 4°C. Alexa-488 conjugated goat anti rabbit (1:200, Invitrogen, USA) and Alexa-594 conjugated donkey anti mouse (1:200, Invitrogen, USA) were used to label the primary antibodies for incubation 40 min at 37°C. Staining SGNs were observed under a fluorescence microscope (Olympus, Japan) and three photos of different groups were taken by microscope.
10
Immunofluorescent Protein Detection
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Tissue sections were prepared as described above followed by incubation (3 h at 37 °C and ON at 4 °C) with mouse anti-p-Akt (cat # 4051) and rabbit polyclonal anti-caspase-3 primary antibodies, 1:150 in blocking solution (Cell Signaling Technology, cat # 9662, MA, USA). After washing, the slides were incubated (37 °C for 30 min) with Cy3 conjugated goat anti-mouse (for p-Akt) and Alexa 488 conjugated goat anti-rabbit (Invitrogen, cat # A-11008, Carlsbad, CA, USA) secondary antibodies for caspase-3. The procedure was then followed as described above.
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