For male sterility experiments, littermate male mice were euthanized, and their cauda epididymis collected. Otherwise, sperm were collected by incubating a lacerated cauda epididymis in a pre-warmed HEPES-0.1% BSA buffer consisting of 130 mM NaCl, 4 mM KCl, 14 mM fructose, 10 mM HEPES, 1.35 mM CaCl2, 1 mM MgCl2 in a droplet covered by embryo tested neat mineral oil. After incubating the cauda epididymis at 36°C for 30 min to allow the sperm cells to swim out, they were used for the following downstream processes. To take videos of the sperm cells, they remained in the buffer described above while being videoed using a Leica DM IRE2 microscope with the Leica MC170 HD camera. To stain the above sperm cells for a morphological and quantitative viability analysis, eosin-nigrosin staining was performed by using two parts 1% eosin Y (Sigma-Aldrich) and two parts 10% nigrosin (Sigma-Aldrich) well-mixed with one part mouse sperm cells. The resulting mix was then smeared on slides, and a coverslip applied with Cytoseal mounting media. Photographs of these slides were taken using a Nikon C2 DS-Ri1 color camera and analyzed using NIS Elements Viewer. For IVF experiments, a modified version of the Nakagata method was followed,57 and developing embryos were quantified and imaged using a Leica DM IRE2 microscope with the Leica MC170 HD camera.
Leica dm ire2 microscope
Manufactured by Leica camera
Sourced in Germany
The Leica DM IRE2 is an inverted research microscope designed for transmitted light and fluorescence microscopy. It features a stable and ergonomic design, high-performance optics, and a wide range of accessories to support various imaging techniques.
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Lab products found in correlation
Leica ccd camera, by Leica camera (3 mentions)
Qwin plus v3, by Leica camera (2 mentions)
Leica dfc350 fx camera, by Leica camera (2 mentions)
Ab95363, by Abcam (1 mentions)
Dfc420, by Leica camera (1 mentions)
Image pro plus v6, by Media Cybernetics (1 mentions)
Dfc350 fx camera, by Leica camera (1 mentions)
Chambered coverglass, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Lsm 710 nlo confocal microscope, by Zeiss (1 mentions)
Smad4, by Abcam (1 mentions)
Heatr1, by Abcam (1 mentions)
Pab anti γh2ax, by Abcam (1 mentions)
Leica dfc350fx, by Leica camera (1 mentions)
Nigrosin, by Merck Group (1 mentions)
Leica mc170 hd camera, by Leica camera (1 mentions)
Eosin y, by Merck Group (1 mentions)
Qwin plus software, by Leica camera (1 mentions)
Anti mouse igg h l, by Cell Signaling Technology (1 mentions)
Rabbit pab anti trf1 n19, by Santa Cruz Biotechnology (1 mentions)
13 protocols using leica dm ire2 microscope
1
Sperm Collection and Viability Analysis for Male Sterility
2
Tissue Microarray Immunohistochemistry Protocol
3
Dual Immunolabeling of FosB/ΔFosB and p-ERK1/2 in CA1 Neurons
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Sections were washed thrice in PBS at room temperature and permeabilized for 1 h in PBS/0.1% Triton X-100 containing 5% normal goat serum. For double immunolabeling, sections were incubated overnight with rabbit polyclonal anti-FosB/ΔFosB (1:250) and mouse monoclonal anti-p-ERK1/2 (1:500) antibodies. After washing, sections were incubated for 3 h at room temperature with secondary antibodies Alexa Fluor 488-labeled goat anti-rabbit (A-11001; Invitrogen, Carlsbad, CA, USA; 1:500) and Alexa Fluor 594-labeled goat anti-mouse antibody (A-11005; Invitrogen, Carlsbad, CA, USA; 1:500). Sections were mounted and coverslipped with Mowiol. Images were acquired using a Leica TCS SP2 (Leica Laser technik, Heidelberg, Germany) confocal multiband scanning laser equipment with AOBS system adapted to an inverted Leica DM IRE2 microscope interfaced with an Argon-Kripton laser set at a power of 8 mW in each line (488, 568) and operating in the single scan acquisition mode. To minimize the noise and to keep a low photobleaching rate, we selected an acquisition time of 2 s per scan and averaged two scans to produce each 1024 × 1024 pixel image. All images of fluorescent neurons in CA1 were taken at HCX PL APO 40× magnification and the final images were the merge of a rostro-caudal series of 4–6 images in a depth of 4–6 μm. The number of double-labeled cells were counted using Fiji software2 .
4
Tissue Microarray Analysis of Glypican-3, Erk1/2, and AKT1
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A total of 229 specimens were selected, and the representative core of each specimen was utilized to construct tissue microarrays. IHC was carried out and samples were measured according to previous reports (36 (link), 37 (link)). The system for imaging included a CCD camera by Leica, DFC420, linked to a Leica DM IRE2 microscope obtained from Leica Microsystems Imaging Solutions, Cambridge, UK. The representative field images were taken from individual core under 200×magnification employing Leica QW in Plus v3 software. Counting and measurement of the photographs IOD were carried out with software of Image-Pro plus V6.0 (Media Cybernetics, Bethesda, MD, USA), and the parameters used were IOD and Area sum. Dilution of the Primary antibodies was done as follows: a rabbit monoclonal [SP86] to Glypican 3 (ab95363; Abcam, Hong Kong; 1:100 dilution, cytomembrane staining), a monoclonal rabbit antibody against Erk1/2 (137F5)(4695; Cell Signaling Technology, Danvers, MA, USA; 1:100 dilution, cytoplasmic staining), a rabbit monoclonal antibody against AKT1 (D9R8K)(75692, Cell Signaling Technology, Danvers, MA, USA; 1:200 dilution, cytoplasmic staining).
5
Immunofluorescence Protocol for TRF1 and γH2AX
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Immunofluorescence (IF) was performed as previously described (Salvati et al., 2007 (link)). Briefly, cells were fixed in 2% formaldehyde and permeabilized in PBS plus 0.25% Triton X-100 for 5 min at room temperature. For immunolabeling, cells were incubated with primary antibody for 2 h at room temperature, washed twice in PBS and finally incubated with the secondary antibodies for 1 h. The following antibodies were used: rabbit policlonal anti-TRF1 antibody (Abcam Ltd.; Cambridge UK); mouse monoclonal anti-γH2AX antibody (Upstate, Lake Placid, NY); TRITC-conjugated Goat anti-Rabbit, FITC-conjugated Goat anti Mouse (Jackson Immunoresearch, Suffolk, UK). Nuclei were immunostained with DAPI. Fluorescence signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by Leica FW4000 deconvolution software (Leica, Solms, Germany). For quantitative analysis of γH2AX positivity, 200 cells on triplicate slices were scored. For TIF analysis, a single plane was analyzed and 30 γH2AX-positive cells were scored. Cells with at least 4 co-localizations (γH2AX /TRF1) were considered as TIF-positive.
6
Nanoparticle-Mediated Cell Imaging and Etching
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PC-3 cells, which express neuropilin-1, a cell surface receptor for the KCDGRPARPAR peptide30 (link), were incubated in 96-well plates with KCDGRPARPAR-coated CdSe/ZnS QDs (QD concentration: 25 nM) for 90 min at 37 °C. The cells were subjected to epifluorescence imaging with a Leica DMIRE2 microscope (Leica, Wetzlar, Germany) before and after the addition of 1x Ag-TS (1 µl) to each well containing 100 µl of culture media. MCF10CA1a cells were incubated with or without iRGD peptide (final concentration: 50 µM) in culture media in chambered coverglass (Nunc Lab-Tek II, Rochester, NY) for 30 min at 37 °C, and ZHS-QDs were added to each chamber (final concentration: 1 mM Zn2+). After incubation for 2.5 h at 37 °C, the cells were washed once with PBS, and cultured in fresh culture media containing Hoechst 33342 (10 µg ml−1 in 400 µl, Molecular Probes, Eugene, OR) for 10 min at 37 °C. Etching was performed by adding 1x Ag-TS (100 µl) to each chamber and incubating the cells for 1 min at RT. The cells were imaged with a Zeiss LSM 710 NLO confocal microscope (Carl Zeiss, Oberkochen, Germany) before and after etching.
7
Tissue Microarray Staining Protocol
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A tissue microarray was constructed following the standard tissue protocols as described previously [13 (link)]. Primary antibodies were HEATR1 (rabbit monoclonal; 1 : 500; Abcam, USA), ZNF185 (rabbit polyclonal; 1 : 1000: Invitrogen, USA), and SMAD4 (rabbit monoclonal; 1 : 100; Abcam, USA) followed by incubation with the secondary antibody. The positive staining was measured by a computerized image system including a Leica-CCD camera connected to a Leica-DM-IRE2 microscope. Pictures of representative fields were captured by the Leica QWin Plus v3 software. The specimens with negative or weak intensity (±) of HEART1, ZNF185, and SMAD4 were graded as low expression, while those with moderate or strong (++/+++) were graded as high expression.
8
Immunofluorescence Analysis of DNA Damage
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Cells were fixed in 2% formaldehyde and permeabilized in 0.25% Triton X100 in PBS for 5 min at room temperature. For immunolabeling, cells were incubated with primary antibody, then washed in PBS and incubated with the secondary antibodies. The following primary antibodies were used: pAb and mAb anti-TRF1(Abcam Ltd.; Cambridge UK); mAb (Upstate, Lake Placid, NY) and pAb anti-γH2AX (Abcam); mAb anti-PCNA (Sigma Chemicals, Milano, Italy). The following secondary antibody were used: TRITC conjugated Goat anti Rabbit, FITC conjugated Goat anti Mouse (Jackson ImmunoResearch Europe Ltd., Suffolk, UK). Fluorescence signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by a Leica FW4000 deconvolution software (Leica, Solms, Germany). This system permits to focus single planes inside the cell generating 3D highresolution images. For quantitative analysis of γH2AX positivity, 200 cells on triplicate slices were scored. For TIFs analysis, in each nucleus a single plane was analyzed and at least 50 nuclei per sample were scored.
9
Tissue Microarray Immunohistochemistry Analysis
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Tissue microarrays were created and stained using standard protocols. A high-sensitivity diaminobenzidine chromogenic substrate system was used for colorimetric visualization. The density of positive staining was measured using a computerized image system and captured using a Leica-CCD camera connected to a Leica-DM-IRE2 microscope (Leica, GER). Under high-power magnification, photographs of representative fields were captured using the Leica Q Win Plus software (version 3). The IHC staining results were evaluated by two independent, experienced pathologists. Finally, according to pAMPK and a-SMA staining, their low and high expression groups were set for comparisons.
10
Quantification of DNA Damage and Telomere Integrity
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Cells were fixed in 2% formaldehyde in phosphate buffered saline (PBS) for 10 min at room temperature (RT) and permeabilized in 0.25% Triton X-100 in PBS for 5 min at RT. For immune-labeling, cells were incubated with primary antibody for 2 h at RT, washed twice in PBS and finally incubated with the secondary antibodies for 1 h. The following primary antibodies were used: Rabbit pAb anti-HMGB1 (Abcam Ltd, Cambridge, UK), Mouse mAb anti-γH2AX (Millipore, Billerica, MA, USA) and Rabbit pAb anti-TRF1 N19 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The following secondary antibodies were used: Anti-Mouse IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 488 Conjugate) (Cell Signaling) and Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 555 Conjugate) (Cell Signaling). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Fluorescence signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by Leica FW4000 deconvolution software (Leica, Solms, Germany). For quantitative analysis of γH2AX positivity, 300 cells on triplicate slices were scored and for TIF analysis, 30 γH2AX-positive cells were scored. Cells with at least four co-localizations (γH2AX/TRF1) were considered as TIF-positive. Where reported, cells were incubated with the indicated doses of Braco-19 for 24 h.
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