For analysis of osteoclast activity, frozen sections of femora isolated from male newborn Cre and hKO mice (7 µm, n = 4 biological replicates per genotype) were stained using the Acid phosphatase, leukocyte (TRAP) Kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Sections were fixed for 30 s, stained for 2 h at 37 °C, mounted in Kaiser's glycerol gelatin and analyzed by light microscopy (Axiophot fluorescence microscope, Zeiss, Germany). To determine osteoclast numbers, deparaffinized sections (7 µm, n = 4 biological replicates per genotype) were stained for TRAP activity as previously described22 (link). Briefly, sections were incubated in 50 mM sodium l -tartrate, 0.1 M sodium acetate, 1.6 mM Fast Red Violet LB salt (Sigma-Aldrich, USA), 0.3 mM Naphthol AS-MX phosphate (Sigma-Aldrich, USA) and 0.5% (v/v) 2-ethoxyethanol (Sigma-Aldrich, USA), pH 5 for 45 min at 37 °C, counterstained with 0.02% (w/v) Fast Green for 30 s, mounted in Kaiser's glycerol gelatin and analyzed by light microscopy (Axiophot fluorescence microscope, Zeiss, Germany). For quantification, the number of multinucleated cells per mm trabecular bone was calculated.
Axiophot fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Axiophot fluorescence microscope is a high-performance optical instrument designed for advanced microscopy applications. It features a sturdy, ergonomic design and delivers excellent image quality. The Axiophot is capable of fluorescence imaging, providing researchers with a powerful tool for a variety of scientific investigations.
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Lab products found in correlation
Anti dig antibodies ap fragments, by Roche (2 mentions)
Nbt bcip for 6 18h, by Merck Group (2 mentions)
Lsm 510 confocal laser scanning microscope, by Zeiss (2 mentions)
Anti dnp pod, by Roche (2 mentions)
A1 confocal microscope, by Zeiss (2 mentions)
Tissue tek oct compound, by Electron Microscopy Sciences (2 mentions)
Sc 20466, by Santa Cruz Biotechnology (2 mentions)
Vectashield mounting media with dapi, by Vector Laboratories (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Amplex red cholesterol assay kit, by Thermo Fisher Scientific (2 mentions)
Glutathione assay kit, by Merck Group (2 mentions)
Filipin 3, by Merck Group (2 mentions)
Axiovision software, by Zeiss (2 mentions)
Axiocam mrm camera, by Zeiss (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Tcs spe confocal microscope, by Leica (2 mentions)
In situ cell death detection kit, by Roche (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor 555 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
64 protocols using axiophot fluorescence microscope
1
Quantifying Osteoclast Activity in Newborn Mice
2
Quantifying Dopaminergic Neurons in Rat SNpc
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Rats were deeply anesthetized by isoflurane and transcardially perfused with 4% paraformaldehyde. Coronal brain sections 20-μm-thick containing the SNpc were washed in 0.1 M phosphate-buffered solution (PBS) contained 0.3% Triton, placed in blocking buffer containing 3% goat serum for 2 hours, and then probed with rabbit anti-tyrosine hydroxylase (TH) antibody (1:1000; Cat# AB152; Millipore, Bedford, MA, USA) to label DA neurons. Sections were labeled with primary antibodies at 4°C overnight, rinsed three times with 0.3% Triton in 0.1 M PBS, and subsequently exposed for 2 hours to goat anti-rabbit IgG (H+L) Alexa Fluor 555 (1:500; Cat# A-21428; Invitrogen, Eugene, OR, USA) at room temperature. Sections were rinsed in PBS and fixed using 70% glycerol. The fluorescent signals were acquired using an Axiophot fluorescence microscope (Zeiss, Oberkochen, Germany). Image Pro Plus software (Media Cybernetics Inc., Rockville, MD, USA) was used to count TH neurons in the SN from every section at 100× magnification. The substantia nigra of each rat was diricled into eight sets of parallel sections, and the total number of neurons in each brain was calcalated by adding the neurons in the eight sets of sections. The number of total TH neurons in each brain was obtained by multiplying the counts by eight sections.
3
Histological Analysis of Infected Mouse Lungs
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Mice were euthanized at the indicated day postinfection (dpi) or day postchallenge (dpc). The left lungs of infected mice were fixed in 10% zinc formalin for 24 h at 4 °C and paraffin embedded. Serial longitudinal 5-μm sections were stained with hematoxylin and eosin by the Histology Service at CNB-CSIC (Madrid, Spain) and subjected to histopathological examination with a ZEISS Axiophot fluorescence microscope. Samples were obtained using a systematic uniform random procedure, consisting of serial parallel slices made at a constant thickness interval of 50 μm. Histopathology analysis was conducted in a blind manner by acquiring images of 50 random microscopy fields from around 40 nonadjacent sections for each of the three independent mice analyzed per treatment group.
4
In Situ Hybridization of Developmental Regulators
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Expression of dlx1a, dlx2a, dlx5a, dlx6a and gad65 was determined using In situ hybridization assays with antisense mRNA probes on crysections as described (Dorsky et al., 1995). Antisense mRNA probes were labeled with digoxygenin-dNTP or dinitrophenol DNP-11-UTP and synthesized from cDNA clones, dlx1a [6 (link)], dlx2a and dlx5a [5 (link)], dlx6a [24 (link)] and gad65 [25 (link)]. Vectors containing the cDNA clones were linearized with BamHI, EcoRI or Xhol and the antisense riboprobes were synthesized using either the T7 or T3 polymerase as required.
Brain sections, stored at -20°C, were thawed at room temperature for 30 minutes before the experiment. Hybridization was carried out overnight at 70°C in a humidified chamber. Slides were washed twice with Solution A (50% Formamide, 5% 20x SSC in dH20) and twice with TBS. Blocking with 10% FBS TBST was carried for 2 hours in RT. Detection of hybridized probes was performed with anti-DIG antibodies AP fragments (Roche, Basel Switzerland; dilution 1:1000) overnight at 4°C. After four TBST washes, staining was developed with NBT/BCIP for 6–18h (Sigma, St-Louis, MO). Images were acquired with a Zeiss AxioPhot Fluorescence Microscope.
Brain sections, stored at -20°C, were thawed at room temperature for 30 minutes before the experiment. Hybridization was carried out overnight at 70°C in a humidified chamber. Slides were washed twice with Solution A (50% Formamide, 5% 20x SSC in dH20) and twice with TBS. Blocking with 10% FBS TBST was carried for 2 hours in RT. Detection of hybridized probes was performed with anti-DIG antibodies AP fragments (Roche, Basel Switzerland; dilution 1:1000) overnight at 4°C. After four TBST washes, staining was developed with NBT/BCIP for 6–18h (Sigma, St-Louis, MO). Images were acquired with a Zeiss AxioPhot Fluorescence Microscope.
5
Immunolocalization of Transglutaminase in Plant Pistils
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Compatible and self-incompatible pistils were directly thawed in a buffer solution (100 mM Pipes pH 6.8, 10 mM EGTA, 10 mM MgCl2, 0.1% NaN3) plus detergent and fixative (0.05% Triton X-100, 1.5% paraformaldehyde, 0.05% glutaraldehyde) for 30 min on ice and then at 4°C for an additional 30 min. For the localization of TGase, fixed pistils were cut along their length and placed in the buffer solution containing 0.75% cellulysin and 0.75% pectinase for 7 min. For immunofluorescence microscopy, samples were washed in the above buffer and incubated with the anti-TGase antibody Ab3 (Neomarker, Fremont, CA, USA) diluted 1:20 in the buffer; incubation was 1 h at 37°C according to previous literature (Del Duca et al., 2009 (link)). After washing with buffer, pistils were incubated with the Alexa-Fluor 488-conjugated goat anti-mouse (Thermo Fisher Scientific) secondary antibody diluted 1:50 in the buffer solution, for 45 min at 37°C in the dark. Samples were observed with a Zeiss Axiophot fluorescence microscope (Ex-Max 490 nm/Em-Max 525 nm) equipped with a MRm video camera and a 63× oil-immersion objective.
6
Quantitative Imaging of Tumor Microenvironment
7
Dual-Antibody Immunostaining for DIG and DNP
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Sections were treated with 2% H2O2 in PBS to inactivate endogenous peroxidase followed by incubation with anti-DIG antibodies POD fragments in combination with anti-DNP POD (Roche, Basel Switzerland; dilution 1:1000). Incubations with these antibodies were done separately at 4°C, overnight, for each of the antibodies. Staining with tyramide Cy3 solution or Fluorescein in PBS/Tween (1:100) was carried for 10 min each (Perkin-Elmer, Woodbridge, Ontario). Images were acquired with a Nikon A1 confocal microscope and/or Zeiss AxioPhot Fluorescence Microscope and treated with NIS-Elements Advanced Research Software or ImageJ.
8
Immunofluorescence Staining of Transfected Cells
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Transfected and non-transfected cells were grown on glass cover slips in 24-well dishes. After 3 days of transfection, the cells were fixed with 2% paraformaldehyde and permeabilized using 0.2% Triton X-100 in PBS. Cells were blocked with 10% normal goat serum in PBS after three washes with PBS. Primary antibodies were added at a dilution of 1:1000 and incubated for 60 min, followed by washings to remove unbound antibodies. Bound primary antibodies were detected with a secondary fluorescent-labelled antibody for a further 60 min. Antibodies directed against protein disulfide isomerase (PDI) (Biomol, Hamburg, Germany) and the 58K protein Sigma (Munich, Germany) were used as markers for the ER and the Golgi apparatus, respectively. Mouse anti-myc antibodies were used to detect the myc epitope of recombinantly-expressed collagen II. Rabbit anti-BiP was used for the detection of BiP. Secondary anti-rabbit and secondary anti-mouse Cy3- and Alexa488-conjugated antibodies were from Molecular Probes (Leiden, The Netherlands). Nuclei were stained with bisbenzimide (Sigma, 0.1 µg/mL). The slides were finally mounted in DAKO fluorescent mounting medium and examined under an Axiophot fluorescence microscope (Zeiss, Oberkochen, Germany).
9
Fluorescence Microscopy Image Analysis
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Fluorescence was visualized with Zeiss Axiophot fluorescence microscope and analysis of images was performed using ImageJ version 1.43u.
10
Fluorescence Microscopy Image Analysis
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Fluorescence was visualized with Zeiss Axiophot fluorescence microscope and analysis of images was performed using ImageJ version 1.43u.
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