Journal: Journal of visualized experiments : JoVE

Article Title: Non-Invasive Compression-Induced ACL Injury and In Vivo Imaging of Protease Activity in Mice

doi: 10.3791/65249

Figure Lengend Snippet: Table of Materials

Article Snippet: Name of Material/ Equipment Company Catalog Number Comments/Description Materials testing systems TA Instruments Electroforce 3200 or equivalent Uniaxial load cell TA Instruments 20 N capacity Fixtures Custom-made knee fixture, ankle fixture, and platform Air Anesthetia System Isoflurane vaporizor with induction chamber and nose cone Buprenorphine Analgesic post-injury IVIS Spectrum Perkin Elmer 124262 Can also use comparable optical imaging system ProSense680 Perkin Elmer NEV10003 Can also use other probes such as OsteoSense, MMPSense, Cat K, AngioSense, etc.

Techniques: Optical Imaging, Cream, Saline, Sterility

Journal: Journal of visualized experiments : JoVE

Article Title: Non-Invasive Compression-Induced ACL Injury and In Vivo Imaging of Protease Activity in Mice

doi: 10.3791/65249

Figure Lengend Snippet: Table of Materials

Article Snippet: ProSense680 , Perkin Elmer , NEV10003 , Can also use other probes such as OsteoSense, MMPSense, Cat K, AngioSense, etc..

Techniques: Optical Imaging, Cream, Saline, Sterility

Journal: Nature Communications

Article Title: In situ and transcriptomic identification of microglia in synapse-rich regions of the developing zebrafish brain

doi: 10.1038/s41467-021-26206-x

Figure Lengend Snippet: a Schematic of analysis pipeline overlaying brain-region-defining genes identified with bulk-sequencing onto mpeg1.1 + scRNA-sequencing clusters. Colors correspond to clusters defined in Fig. . b Feature plots of region-defining eigengenes from hindbrain (HB; red), midbrain (MB; green), and optic tectum (OT; blue) overlaid on mpeg1.1 + UMAP, highlighting microglial clusters JM1 and JM4. Eigengenes were composed of single region marker genes with regional enrichment (log fold change >1.2, basemean >100, and adj. p < 0.05) from the bulk-sequencing analysis in Fig. and computed with the PercentageFeatureSet function in the Seurat R package. Color-coding for Eigengene expression: OT, low expression; light blue, high expression; dark blue. MB, low expression; light green, high expression; dark green. HB, low expression; pink, high expression; red. c Violin plots of regional eigengene distributions, related to b . Colors correspond with clusters shown in Figs. c, 4a. (Solid black line = median; dotted lines = 1st and 3rd quartiles). d Volcano plot of differentially expressed genes between clusters JM1 (OT-enriched; blue) and JM4 (hindbrain-enriched; red). Thresholds represented by dotted lines were set to adjusted p value < 10 −8 , log fold change >0.2. (MAST differential expression test with Bonferroni correction; See Supplemental S5). e Top GO terms from differentially expressed genes in d . (Metascape; hypergeometric test with Benjamini–Hochberg correction for multiple comparisons). f Representative images of cathepsin-cleaved Prosense680 colocalized with mpeg1.1- EGFP in indicated brain regions. Scale: 100 µm. Images are representative of the n = 3 replicates. g Quantification of total percent microglia containing cleaved Prosense680 in OT and HB. Unpaired t tests. Dots represent three individual fish, data are mean ± SD. * p < 0.0236. OT (Optic Tectum) and HB (Hindbrain). Two-tailed unpaired t test. h Representative images and 3D reconstructions of mpeg1.1- EGFP+ microglia with engulfed SV2 protein. Insets: close-up of reconstructions (arrowheads:SV2 content). Scale bar 5 µm. Images are representative of ameboid or ramified morphology. i Quantification of percent microglial volume containing SV2 in randomly selected microglia from OT and HB. Post hoc analyses show morphology assignment as ramified (red, sphericity <0.6) vs. ameboid (blue, sphericity >0.6. Mann–Whitney U test, * p < 0.0177, dots = mean value per fish from three microglia per fish. See also Fig. and Fig. , Supplementary Data .

Article Snippet: To identify our putative OT-enriched cathepsin-rich cluster (JM1) in situ, we quantified functional cathepsin activity with the biomarker Prosense680, which becomes fluorescent after proteolytic cleavage by cathepsins with a preference for cathepsin B and L . Microglial cathepsin activity ( mpeg1.1 -EGFP + Prosense + ) was highly enriched in the OT, where it was mostly detected in ameboid microglia around neurogenic regions (Fig. ), closely matching the expression of ctsba by ISH (Fig. ).

Techniques: Sequencing, Marker, Expressing, Quantitative Proteomics, Two Tailed Test, MANN-WHITNEY