All quantification was carried out by an observer blinded to treatment. Sections were chosen based on the following criteria: Sections (1) contained transplanted TH+ cell bodies and (2) either had visible needle tracts from lentiviral pathway injections or were located in sections between 4 and 8.0 mm ventral to Bregma according to the rat brain atlas.48 Only horizontal sections were used for analyses of axon growth along the pathway. Sections without striatum (too dorsal) or substantia nigra (too ventral) were not included in the analysis. To quantify TH+ axonal growth, the number of identifiable TH+ fibers was counted manually with a 0.5-mm2 grid at 200× at a distance of 2–3 mm from the rostral edge of the transplant (near box E in Figure 3 B) from 6 sections per animal spaced 100 μm apart and an average of axon counts used per rat (5–6 rats per group). For counting the number of cells labeled with TH, GirK2, or calbindin, FG immunofluorescence-treated sections were examined with an Olympus AX80 microscope equipped with fluorescein and UV filters. Sections containing triple-labeled cells were sampled using a confocal microscope, and co-localization was confirmed by z axis analysis. Quantification was performed on 6 evenly spaced sections (80 μm apart) within the transplanted region.
Ax80 microscope
Manufactured by Olympus
Sourced in Japan, United States
The AX80 microscope is a high-performance optical microscope designed for laboratory and research applications. It features a sturdy, ergonomic construction and advanced optical components to provide clear, high-resolution images. The AX80 is capable of various magnification levels to accommodate a wide range of sample types and observation needs.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated anti rabbit igg, by Vector Laboratories (3 mentions)
Abc method, by Vector Laboratories (3 mentions)
Rm2235 radial microtomes, by Leica Microsystems (2 mentions)
Dp70 digital camera, by Olympus (2 mentions)
Biotinylated anti mouse igg, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Dojindo Laboratories (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Mildform, by Fujifilm (1 mentions)
Dako liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
Universal hier antigen retrieval reagent, by Abcam (1 mentions)
Block ace, by Bio-Rad (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Bz x710 microscope, by Keyence (1 mentions)
Matrigel, by BD (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Dp50 digital camera, by Olympus (1 mentions)
Cm3050 cryostat, by Leica camera (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
28 protocols using ax80 microscope
1
Quantification of Neuronal Axon Growth
2
Fluorescence Imaging of Rat Brains and Spinal Cords
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Prior to histological preparation, intact brains and spinal cords from rats that received AAV2/9-shPTEN-zsGreen or AAV2/9-shLuc-zsGreen were examined by fluorescence epi-illumination using an Olympus AX80 microscope; images were taken at 2× and tiled to create a complete image of the brain. For tdT reporter mice that received AAV2-retro/Cre injections into the spinal cord, brains and spinal cords were removed intact. Brains were placed on a glass slide and illuminated by epifluorescence; images were taken at 2× and tiled to create a complete image of the brain and spinal cord.
3
Immunohistochemical Analysis of Renal Tissue
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Immunohistochemistry was performed as previously described [22 (link)]. For immunostaining, mouse tissues were fixed with Mildform (Wako Pure Chemical, Tokyo, Japan). For human renal tumor tissues, a prefixed human tissue array was purchased (KD2082a, US Biomax Inc., Rockville, MD, USA). After paraffinization, the tissues were sectioned and deparaffinized using xylene and ethanol, followed by antigen retrieval using Universal HIER Antigen Retrieval Reagent (Abcam). Samples were subjected to hematoxylin & eosin staining or immunostaining. After blocking with Block ACE (Bio‐Rad, Hercules, CA, USA), the samples were incubated with primary antibodies and the appropriate secondary antibodies (Table S4 ) and stained using a Dako Liquid DAB+ Substrate Chromogen System (Agilent Technologies, Santa Clara, CA, USA) and Mayor’s hematoxylin. Images were captured using an AX80 microscope (Olympus, Tokyo, Japan).
To detect apoptosis, In Situ Cell Death Detection Kit (TMR red; Roche Diagnostics) and DAPI Fluoromount‐G (Southern Biotech, Birmingham, AL, USA) were used as previously described [32 (link)]. Fluorescent images were captured using a BZ‐X710 microscope (KEYENCE, Osaka, Japan).
To detect apoptosis, In Situ Cell Death Detection Kit (TMR red; Roche Diagnostics) and DAPI Fluoromount‐G (Southern Biotech, Birmingham, AL, USA) were used as previously described [32 (link)]. Fluorescent images were captured using a BZ‐X710 microscope (KEYENCE, Osaka, Japan).
4
Microscope Imaging Protocols
5
Teratoma Formation from Reprogrammed iPMSCs
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Reprogrammed iPMSC suspensions (1 × 107) were mixed with matrigel (BD Bioscience) and injected subcutaneously into NOD/SCID mice without anesthesia. After 2 months teratomas were collected and fixed in 4 % paraformaldehyde in PBS, embedded in paraffin, and sectioned at 10 μm using RM2235 radial microtomes (Leica Microsystems). Sections were subjected to histologic staining with H & E and immunohistochemistry staining of HLA-ABC as already described and observed under an AX80 microscope (Olympus).
6
Histological Analysis of Tissue Engineering
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Following paraformaldehyde fixation, the scaffolds were embedded in paraffin. Ten micron tissue sections were cut using RM2235 radial microtomes (Leica Microsystems) for histological analysis and stained with hematoxylin and eosin (H & E). In the osteogenic group, tissue sections were also stained by a modified Masson Trichrome [25 ] and Von Kussa [26 ] staining. Five mice from each group were analyzed. Immunohistochemistry staining of HLA-ABC (CST) at 1:1000 dilution were used to detect whether the tissue formed in vivo originated from human iPMSCs. Sections were observed under an AX80 microscope (Olympus, Tokyo, Japan).
7
Spinal Cord Axon Degeneration Imaging
8
Microscopic Imaging of Biological Specimens
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Whole-mount LRT specimens were photographed on a Leica M165FC microscope equipped with a DFC310FC digital camera and Application Suite software (ver. 3.3.0; Leica Camera AG, Solms, Germany). Histological sections were photographed on an Olympus AX80 microscope equipped with a DP50 digital camera and Studio Lite software (ver. 1.0; Olympus corporation). Some images were merged and trimmed using Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA, USA).
9
Immunohistochemical Analysis of Microglial Activation
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We utilized pentobarbital (90 mg/kg i.p.) to anesthetize the rats, which then were transcardially perfusion with 200 ml saline followed by 500 ml of 4% paraformaldehyde. Brains were dehydrated in 30% sucrose at 4°C and using a Leica CM3050 Cryostat to section coronally into 40-μm-thick slices. Sections were taken from 2.1 to –1.0 mm (striatum) relative to bregma, then stored in cryopreservant for storage (20% glycerol, 2% DMSO in 1XPBS). Sections were blocked with 4% BSA (Sigma-Aldrich) + 0.1% Triton X-100 (Sigma-Aldrich), then incubated with primary antibody (rabbit anti-CD11b 1:1000; Abcam) overnight at 4°C. The next day, sections were stained with secondary antibody, followed by incubation with secondary antibodies conjugated with Alexa Fluor® 488 or 568 (1:500). Digital imagining was carried out on an Olympus AX-80 microscope and attached DP-70 digital camera (Olympus America Inc., Center Valley, PA, United States) using a 40x objective. Three coronal brain sections per animal (between bregma −0.45 and −1.85) were imaged in the right hemisphere at each time point (sham, 1, 2, and 7 days post-dMCAo) (Morrison et al., 2017 ). The regions imaged were in the medial peri-infarct cortex extending 400 μm from the infarct border and in the dorsal striatum underlying the infarct. Therefore, the imaging yielded six digital photomicrographs per animal for analysis.
10
Immunostaining and Imaging of CTCs
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After CTC capture, immunostaining of the CTCs was performed using human CK19 antibodies, as reported previously (5 (link)), and, in two cases, using anti-CD133 antibodies. When using CD133 antibodies, before fixation with 4% paraformaldehyde in PBS, CTCs were incubated with mouse anti-human CD133 antibodies conjugated with Alexa Fluor 615 (#130-113-671; Miltenyi Biotec GmbH), which were diluted 1:50 for 10 min at room temperature in the CTC-chip microfluidic system. After fixation, CTCs were incubated with sheep anti-human CK19 antibodies (#sc-33119; Santa Cruz Biotechnology), which were diluted 1:200 and incubated for 1 h. Then, donkey anti-sheep antibodies conjugated with Alexa Fluor 488 (#1807723; Thermo Fisher Scientific), which were diluted 1:1,000, were incubated for 30 min as secondary antibodies to visualize CTCs. Finally, the chip was covered with VectaShield with DAPI (Vector Laboratories). When preliminarily testing the compatibility of our CTC chips using cell lines, anti-CK19 antibodies and anti-CD45 antibodies were diluted 1:300, and the fluorescence was evaluated. The fluorescence signals were detected under an Olympus AX80 microscope.
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