Immunostaining of frozen sections was done as described previously18 (link),17 (link). Briefly, retinal explants were fixed with 4% paraformaldehyde and treated with 15% and 30% sucrose in this order, and embedded in the optimal cutting temperature compound (Sakura Fineteck). Primarily antibodies used were monoclonal antibodies against active Caspase 3 (Promega), Ki67 (BD Bioscience), HuC/D (Molecular Probes), Chx10 (Exalha Biologicals), NR2E3 (photoreceptor-specific nuclear receptor, PNR) (ppmx), glutamine synthetase (GS) (Millipore), PKC (Merck Millipore), Rhodopsin (Rho4D2, kindly donated by Dr R. S. Molday, The University of British Columbia), and a polyclonal antibody against GFP (Clontech). Nuclei were counterstained with 4′,6-diamidino-2-phnylindole, dihydrochloride (DAPI). Sections were then treated with Alexa-488-, Alexa-594-, or Alexa-680-conjugated appropriate secondary antibodies. Photos were taken under observation using Zeiss Axio Image M1 and Axio Image M2.
Axio image m2
Manufactured by Zeiss
Sourced in Germany, Gabon
The Axio Image M2 is a high-performance microscope system designed for advanced imaging and analysis applications. It features a modular design that allows for customization to meet the specific needs of researchers and laboratories. The core function of the Axio Image M2 is to provide high-quality, reproducible imaging capabilities for a wide range of samples and applications.
Automatically generated - may contain errors
Lab products found in correlation
Bromodeoxyuridine (brdu), by Abcam (2 mentions)
Csf1r, by Merck Group (2 mentions)
Antibody to goat biotin igg, by Vector Laboratories (2 mentions)
Neuronal nuclei (neun), by Merck Group (2 mentions)
Cd11b, by Abcam (2 mentions)
Lsm 700 microscope, by Zeiss (2 mentions)
Alexa fluor 488 or 594, by Thermo Fisher Scientific (2 mentions)
Csf 1, by R&D Systems (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Huc d, by Thermo Fisher Scientific (1 mentions)
Glutamine synthetase, by Merck Group (1 mentions)
Active caspase 3, by Promega (1 mentions)
Axio image m1, by Zeiss (1 mentions)
Osteomeasure system, by OsteoMetrics (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Zen acquisition and analysis software, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
9 protocols using axio image m2
1
Immunostaining of Retinal Explants
2
Immunostaining of Kv11.1 Potassium Channels
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For immunostaining, the cells were fixed in 4% formaldehyde in phosphate buffered saline (PBS) 0.1 M for 10 min. After washing, permeabilization (Triton 0.1% in PBS) for 10 min and blocking buffer (5% goat serum in PBS) for 30 min were performed. Primary antibody (rabbit anti-potassium channel Kv11.1 extracellular (1:100; P0749; Sigma-Aldrich, Saint Louis, MO, USA) was added in blocking buffer and incubated for approximately 2 h at 4 °C. Cells were washed three times in PBS before incubation with goat anti-rabbit antibody conjugated to Alexa Fluor 647 (1:400; BioLegend, San Diego, CA, USA) for 1 h at room temperature (RT) and subsequently washed with PBS. Cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, 1/30,000 in water) for 5 min. Glass coverslips were finally mounted on microscopy glass slides using a polymerizing medium (FluorSaveTM reagent Calbiochem®-Merk, Darmstadt, Germany). For cell fluorescence acquisition, images were acquired with a Zeiss Microscope (Axio Image M2 equipped with XCite 110 LED lamp, Zeiss, Jena, Germany) and images were captured using Zen (Zeiss, Jena, Germany) software.
3
Fluorescence In Situ Hybridization Protocol for Marker Gene Detection
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The specific DNA probes of the selected marker genes were designed and synthesized using GENWIZ (Supplementary Data Table S8 ). The basal root tips were harvested and immediately placed in 4% paraformaldehyde solution for fixation. The hybridization and immunological methods were performed as described previously [47 (link), 48 (link)]. The images were collected using a Zeiss microscope (Axio Image.M2).
4
Immunostaining of Neuronal Cell Types
5
Immunostaining of Neuronal Cell Types
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We immunostained tissue as previously described53 (link), with the following antibodies: GFP (abcam), CSF1R (Millipore), CD11b (Abcam), ATF3 (Santa Cruz), CSF1 (R&D), NPY (gift from Clark J. Allen), Iba1 (Wako), NeuN (Millipore), BrdU (Abcam), and fluorophore coupled secondary antibodies (Alexa Fluor 488, 555, 594, 647) (Invitrogen). To localize CSF1 in DRG neurons and in their processes, we used antibody to goat biotin IgG (Vector Laboratories) and streptavidin coupled to an Alexa Fluor 488 or 594 (Invitrogen). Images were collected with a Carl Zeiss LSM 700 microscope or a Zeiss Axio Image M2 (DRG overview images only) and were processed with Fiji/ImageJ (NIH). Corresponding images (e.g. ipsilateral vs. contralateral; CSF1 vs. PBS; wt vs. mutant) were processed in an identical manner. Each experiment was performed in at least 3 animals.
6
Paraffin Sectioning for Basal Root Tissues
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The paraffin section method for basal root tissues was described previously [52 (link)]. The staining results were imaged on a Zeiss microscope (Axio Image.M2).
7
Quantitative Histomorphometric Analysis of Bone
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Lumbar vertebrae were fixed for 24 hr in 10% Millonig’s formalin, dehydrated into 100% ethanol, embedded in methyl methacrylate, and then 5 μm longitudinal sections were obtained. After removal of plastic and rehydration, we stained sections for TRAP activity and counter-stained with T-blue. Quantitative histomorphometry was performed to determine osteoblast and osteoclast number using Osteomeasure system (OsteoMetrics, Decatur, GA) interfaced to an Axio image M2 (Carl Zeiss, NY). Bone formation rate was measured using unstained sections in Osteomeasure system. We used terminology recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (Dempster et al., 2013 (link)). For quantification of periosteal and endocortical bone formation, femurs or tibiae were fixed in 10% Millonig’s formalin for 24 hr, dehydrated into 100% ethanol, embedded in methyl methacrylate, and then 80 μm cross sections were obtained at the femoral mid-diaphysis for femoral sections and 5 mm proximal from the distal tibiofibular junction for tibial sections. We then measured mineralizing surface and mineral apposition rate using the Osteomeasure system.
8
Immunostaining of Neurons and Brain Tissue
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Immunostaining of cultured neurons and 100‐μm‐thick brain sections was performed as described 30 . Immunofluorescent images of cultured neurons were visualized at room temperature with a fluorescence microscope (Axioimage M2; Zeiss) equipped with a 20×/NA 0.80 (Plan‐Apochromat) objective lens and acquired using a cooled charge‐coupled device camera (Rolera EM‐C2; QImaging) with Zen software (Zeiss). For brain sections, neuronal images were captured at room temperature with a confocal microscope (LSM 700, Zeiss) equipped with a 20×/NA 0.80 (Plan‐Apochromat) objective lens and Zen acquisition and analysis software (Zeiss). The images were processed using Photoshop (Adobe) with minimal adjustment of brightness or contrast applied to the entire images. The camera lucida drawings were performed with ImageJ.
9
Immunohistochemical Analysis of Microvascular Area
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Tissues were embedded in OCT and snap frozen in liquid nitrogen or xed in 10% buffered formalin followed by para n embedding. IHC staining was applied to 3-µm thick sections of para n-embedded tissue specimens with a PV-9001 Detection Kit (ZSGB-BIO). Brie y, formalin-xed para n-embedded tissues were depara nized in xylene for 10min and rehydrated in a graded series of ethanol solutions. The tissues were immersed in 0.01M citric acid buffer at 121℃ for 5min and then cooled and washed with 0.1M PBS for 3 times. After treated with 3% hydrogen peroxide for 10min, the tissues were incubated with primary antibodies overnight at 4℃, followed by the secondary antibody for 30min at 25℃. Sections were stained with 3,3'-diaminobenzidine and counterstained using hematoxylin for 5 seconds, dehydrated in a graded series of ethanol solutions, immersed in xylene, and examined with a microscope (Axio Image M2, Zeiss). The microvascular area (MVA) in IHC stained images of each group was analyzed with Image-Pro Plus software.
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