The in vitro invasion assays were carried out as described previously [47 (link)]. The Matrigel invasion chambers were prepared at 1:2 dilution of Matrigel (BD Biosciences, Belford, MA) as described before [34 (link)]. Equal numbers of viable cells (4 × 104) were seeded on top of the Matrigel layer. After incubation for 24 h at 37°C, non-invading cells in the Matrigel layer were quantitatively removed, and the microporous membrane containing cells that migrated across the Matrigel layer and through the microporous membrane was stained and viewed under an Olympus BX 61 bright field microscope as described before [34 (link)]. At least six fields were examined within any one experiment for each condition and a representative image for each cell line is presented.
Bx61 bright field microscope
Manufactured by Olympus
Sourced in Japan
The BX61 is a bright-field microscope designed for a variety of laboratory applications. It features a sturdy and ergonomic design, providing a stable platform for precise observations. The BX61 offers high-quality optics, enabling clear and detailed imaging of samples.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (1 mentions)
Matrigel, by Olympus (1 mentions)
Neutral buffered formalin, by Thermo Fisher Scientific (1 mentions)
Ta cloning kit dual promoter, by Thermo Fisher Scientific (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Tissue freezing medium tfmtm, by Electron Microscopy Sciences (1 mentions)
Imagej, by Olympus (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Rabbit anti iba1 antibody, by Fujifilm (1 mentions)
Diaminobenzidine dab, by Vector Laboratories (1 mentions)
Avidin biotin horseradish peroxidase complex, by Vector Laboratories (1 mentions)
7 protocols using bx61 bright field microscope
1
In Vitro Matrigel Invasion Assay
2
Histological Analysis of Reproductive Tract
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The reproductive tract (vagina, cervix, and uterine horns) was harvested as an intact structure, fixed overnight in 10% neutral-buffered formalin (Fisher, NH, USA), paraffin-embedded and sectioned. Slides were stained with hematoxylin and eosin, then imaged with an Olympus (Tokyo, Japan) BX61 bright field microscope. An observer blinded to the infection status of the samples scored the level of polymorphonuclear cells in the tissue based on the following rubric: 0 –absent, 1 –minimal, 2 –moderate, 3 –profound, 4 –severe (including immune cells crossing the epithelium into the lumen).
3
Cloning and Visualization of eaat2a in Zebrafish
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eaat2a (ENSDARG00000102453) cloning into the TOPO pCRII vector (TA Cloning Kit Dual Promoter, Invitrogen, Basel, Switzerland) and preparation of digoxigenin (DIG)‐labeled antisense RNA probes is described elsewhere (Gesemann et al., 2010 (link); Niklaus et al., 2017 (link)). RNA probes were applied on whole‐mount zebrafish larvae (3 and 5 days post fertilization [dpf]) and adult (older than 6 months) brain cross sections at a concentration of 2 ng/μl at 64°C overnight (Huang et al., 2012 (link)). For larval brain sections, representative stained and paraformaldehyde (PFA) post‐fixed embryos were cryoprotected in 30% sucrose at 4°C overnight, embedded in Tissue Freezing Medium TFMTM (Electron Microscopy Sciences), cryo‐sectioned at 14–16 μm and mounted onto Superfrost slides (Thermo Fisher Scientific). PFA post‐fixed whole‐mount embryos (in glycerol) and sections were imaged with an Olympus BX61 brightfield microscope. Images were adjusted for brightness and contrast using Affinity Photo Version 1.8 and assembled in Affinity Designer Version 1.7.
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eaat2a (ENSDARG00000102453) cloning into the TOPO pCRII vector (TA Cloning Kit Dual Promoter, Invitrogen, Basel, Switzerland) and preparation of digoxigenin (DIG)‐labeled antisense RNA probes is described elsewhere (Gesemann et al., 2010 (link); Niklaus et al., 2017 (link)). RNA probes were applied on whole‐mount zebrafish larvae (3 and 5 days post fertilization [dpf]) and adult (older than 6 months) brain cross sections at a concentration of 2 ng/μl at 64°C overnight (Huang et al., 2012 (link)). For larval brain sections, representative stained and paraformaldehyde (PFA) post‐fixed embryos were cryoprotected in 30% sucrose at 4°C overnight, embedded in Tissue Freezing Medium TFMTM (Electron Microscopy Sciences), cryo‐sectioned at 14–16 μm and mounted onto Superfrost slides (Thermo Fisher Scientific). PFA post‐fixed whole‐mount embryos (in glycerol) and sections were imaged with an Olympus BX61 brightfield microscope. Images were adjusted for brightness and contrast using Affinity Photo Version 1.8 and assembled in Affinity Designer Version 1.7.
4
Microglial Morphology Analysis in Hippocampus
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Morphological analyses of microglia were performed by a condition-blind observer using FIJI. The cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG) subfields from the hippocampus were analyzed between Bregma −2.2 and −2.6 mm 20 μm-thick Z-stacked photomicrographs were acquired from an Olympus BX61 bright-field microscope at 1 μm intervals (20 images). This depth allows for complete reconstruction of the microglial soma and processes while avoiding overlap of microglia that would confound our analyses (Fernandez-Arjona et al., 2017 (link)). All analyses had four to seven mice per experimental group.
5
Quantifying Muscle Inflammation via H&E Staining
6
Golgi-Cox Staining of Dendritic Spines
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The Golgi-Cox staining was performed as described previously.47 (link),48 (link) Briefly, the Golgi-Cox solution (1% K2Cr2O7, 1% HgCl2, 0.8% K₂CrO₄) was prepared before its use. Mice were deeply anesthetized with sodium thiopental and decapitated. The brain hemispheres were immersed in the Golgi-Cox solution during three weeks followed by incubation with 90% ethanol for 30 minutes. Coronal brain sections of 200μm thickness obtained in a vibratome were incubated with 15% ammonia and 1% thiosulfate. Brain sections were dehydrated in an ethanol-isopropanol-xylol sequence and mounted in Superfrost-Plus slides with DPX mounting media. Z-stacks images of a 0.5 μm step size containing the full dendritic arbor of CA1 stained neurons were acquired in a BX-61 bright-field microscope (Olympus) with a 100x objective. The number of dendritic spines was manually counted in a known length of secondary dendritic segments using the Extended Depth of Field plugin for ImageJ software (ImageJ, RRID: SCR_003070 ).49 (link),50 (link) The density of dendritic spines was calculated for each dendritic segment and averaged for each experimental group.
7
Immunohistochemical Detection of Microglia
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Prior to immunohistochemistry, slides were blocked with 0.2% Triton X-100 in phosphate-buffered saline containing 10% normal goat serum for one hour at room temperature. Slides were incubated overnight with rabbit anti-Iba1 antibody (1:1,000; Wako; RRID: AB_839504) in blocking buffer at 4 °C, after which they were incubated in biotinylated secondary antibody (1:1,000; Vector Laboratories; Catalog Number: BA-1000-1.5) for one hour and avidin–biotin horseradish peroxidase complex (Vector Laboratories; Catalog Number: PK-6100) for another hour. The sections were then incubated in diaminobenzidine (DAB; Vector Laboratories; Catalog Number: SK-4100) to visualize the activity of the horseradish peroxidase. This reaction was terminated after three minutes when the contrast between the microglia and nonspecific background labeling was optimal. The sections were then dehydrated in a series of alcohols, cleared in xylene, and coverslipped with Permount. Images were captured using an Olympus BX61 bright-field microscope.
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