Journal: PLoS Pathogens

Article Title: Type I Interferons Direct Gammaherpesvirus Host Colonization

doi: 10.1371/journal.ppat.1005654

Figure Lengend Snippet: a. Mx1-cre mice were given MHV-4-RG i.n. (3x10 4 p.f.u. in 30μl under anesthesia). Viruses were recovered from lungs by plaque assay and from lymphoid tissue by intact cell explant onto BHK-21 cell monolayers. Plaques were then typed as eGFP + (switched) or mCherry + (unswitched). For each mouse (circles), % eGFP + = % switched of total recovered plaques. Crosses show means. MLN = mediastinal lymph nodes. b. Mice were given MHV-RG i.n. (3x10 4 p.f.u. in 5μl without anesthesia) to infect just the nose, then analysed for viral fluorochrome switching as in a . SCLN = superficial cervical lymph nodes. Viruses were recovered from noses by plaque assay and from SCLN by intact cell explant (infectious centre assay). c. Mice were given i.n. or i.p. MHV-RG, with or without poly(I:C) (50μg i.n. or i.p. 6h before and at the time of infection) to maximally induce IFN-I. Viruses recovered 3d later from lungs by plaque assay (i.n.) or from spleens by infectious centre assay (i.p.) were analysed for fluorochrome expression as in a . d. Mice were infected i.n. (lung) or i.p. (spleen) and given poly(I:C) or not as in c . Virus titers were determined 3d later by plaque assay. Circles show individuals, crosses show means. e. Mx1-cre mice were depleted or not of pDCs with mAb 120G8, then infected i.p. with MuHV-RG (10 5 p.f.u.). 5d later spleens were titered for total recoverable virus by explant of intact splenocytes onto BHK-21 cells. Infectious centres (ICs) were also typed as mCherry + (unswitched) or GFP + (switched). % eGFP + = % of total plaques that were switched. Bars show mean ± SEM, other symbols show individual mice. pDC depletion had no significant effect.

Article Snippet: Sections (6μm) were air-dried (1h, 23°C), washed 3x in PBS, blocked with 0.3% Triton X-100 / 5% normal donkey serum (1h, 23°C), then incubated (18h, 4°C) with combinations of antibodies to eGFP (rabbit, chicken or goat pAb), CD68 (rat mAb, FA-11) (AbCam), B220 (rat mAb RA3-6B2), F4/80 (rat mAb CI:A3–1) (Santa Cruz Biotechnology), mCherry (rabbit pAb, Badrilla), CD206 (rat mAb MR5D3), CD169 (rat mAb 3D6.112) (Serotec), podoplanin (goat pAb, R&D Systems), and MuHV-4 (polyclonal rabbit sera raised by 2 subcutaneous virus inoculations).

Techniques: Plaque Assay, Infection, Expressing

Journal: PLoS Pathogens

Article Title: Type I Interferons Direct Gammaherpesvirus Host Colonization

doi: 10.1371/journal.ppat.1005654

Figure Lengend Snippet: a. Mx1-cre mice were given i.n. poly(I:C) (50μg) to induce IFN-I 6h before and at the time of i.n. infection with MHV-RG (3x10 4 p.f.u.). 5d later lung sections were stained for viral eGFP / mCherry, and the cell markers podoplanin (PDP, AEC1s), CD68 (macrophages) and CD169 (AMs). Each image is representative of 5 mice. Arrows show example positive cells. Quantitation is shown in b . The cytoplasmic rather than nuclear distribution of eGFP is typical for the relatively mild fixation of periodate-lysine-1% formaldehyde. b. Lung sections of mice infected as in a (5 per group) were analysed at d4-6, counting eGFP + (green) and mCherry + (red) PDP + and CD68 + cells for 3 sections per mouse. Each section corresponds to approximately 10 fields of view as illustrated in a . Bars show mean ± SEM. At d5 CD68 + cells showed significantly more fluorochrome switching than PDP + cells (p<0.01). c. Mx1-cre mice were infected i.p. with MHV-RG (10 5 pfu), and eGFP + (green) and mCherry + (red) MZ macrophages (CD169 + ) and B cells (B220 + ) counted 4d later. Bars show mean ± SEM counts for 3 sections each of 3 mice per group. B220 + cells were significantly more switched than unswitched, whereas CD169 + were significantly more unswitched than switched (p<0.02). d. Example images of mice infected as in c . Arrows show positive cells. e. Mx1-cre mice were infected i.p. with MHV-RG (10 5 pfu), as in d , but poly(I:C) (50μg i.p.) was given 6h before and at the time of infection to test the effect of increased IFN-I induction. Spleen sections were analysed at d4 for viral eGFP and mCherry expression in B220 + B cells and CD68 + CD169 + MZ macrophages. Arrows show example positive cells. Quantitation is shown in f . f. Mean ± SEM counts are shown for 3 sections each of 5 mice per group, analysed as in e at d3-5 post-infection. Each field of view was 10x the area of the images shown in e , and we counted at least 10 fields of view per section (at least 200 viral fluorochrome + cells). B220 + cells showed significantly more switched than unswitched fluorochrome expression at d3 and d4 of infection (p<10 −4 ). CD68 + cells did not (p>0.5). g. Mice were infected as in e and infected MZ B cells identified by staining for IgM. WP B cells express lower levels of IgM and while this is detectable by flow cytometry, by confocal microscopy as used here, WP B cells are IgD + IgM - . >90% of infected MZ B cells were eGFP + , confirming the results of e-f .

Article Snippet: Sections (6μm) were air-dried (1h, 23°C), washed 3x in PBS, blocked with 0.3% Triton X-100 / 5% normal donkey serum (1h, 23°C), then incubated (18h, 4°C) with combinations of antibodies to eGFP (rabbit, chicken or goat pAb), CD68 (rat mAb, FA-11) (AbCam), B220 (rat mAb RA3-6B2), F4/80 (rat mAb CI:A3–1) (Santa Cruz Biotechnology), mCherry (rabbit pAb, Badrilla), CD206 (rat mAb MR5D3), CD169 (rat mAb 3D6.112) (Serotec), podoplanin (goat pAb, R&D Systems), and MuHV-4 (polyclonal rabbit sera raised by 2 subcutaneous virus inoculations).

Techniques: Infection, Staining, Quantitation Assay, Expressing, Flow Cytometry, Confocal Microscopy

Journal: Frontiers in Cellular Neuroscience

Article Title: Quantitative characterisation of ipRGCs in retinal degeneration using a computation platform for extracting and reconstructing single neurons in 3D from a multi-colour labeled population

doi: 10.3389/fncel.2022.1009321

Figure Lengend Snippet: Identifying and isolating voxel clusters from multi-color images in Principal Component Space. (A) Following a unilateral intravitreal injection of Brainbow viruses into an Opn4 Cre/+ mouse, immunohistochemical labeling against the mCherry reporter protein (in monochrome) demonstrates strong labeling of cells distributed across the retinal ganglion cell layer. (B) Maximum projection of the immunohistochemically labeled mCherry, mTFP eYFP and tagBFP reporter proteins from a region of interest (ROI) from an Opn4 Cre/+ retina demonstrates variable reporter protein expression between individual intrinsically photosensitive retinal ganglion cells (ipRGCs) in the Opn4 Cre/+ retina. (Note weak expression of tagBFP and high background noise) (C) Representative confocal image of the ROI shows stochastic labeling of individual cells when the combined maximal projection of the three individually recorded channels mCherry (pseudocolored Red), eYFP (pseudocolored Green) and mTFP (pseudocolored Blue) from panel (B) , following immunohistochemical labeling. (D) Principal component analysis (PCA) conducted on the intensity values of the constituent voxels of ROI in panel (B) were plotted in a 3D representation of PC space (PC1 vs. PC2 vs. PC3). (E,Ei) Simple simulated image of four colored bars, with hue and saturation kept constant across each bar, but with lightness varying from 0 (black) at the outer edge to either 0.6 (in the case of purple and orange) or 0.9 (in the case of cyan and gray) in the center (see online methods and for further explanation). (Eii) 3D representation of PC space from the image in panel (Ei) shows that the cyan cluster of voxels diverging from the black point, reaches a point of inflection, and tends toward the white point. (Eiii) Extraction of the cyan cluster (from black point to the white point via the inflection point) in 3 × 2D representations of PC space (PC1 vs PC2, PC1 vs. PC3 and PC2 vs. PC3) reconstructs the entirety of the cyan bar (Eiv) . For more details see and codes are available for this Figure at: https://github.com/lucasgroup/BRIAN (BBStatic.py) (F,Fi) Maximum projection of a ROI of an optic nerve from Opn4 Cre/+ ;rd/rd mouse unilaterally intravitreally injected with the AAV Brainbow virus, following immunohistochemical labeling for mCherry (pseudocolored red), eYFP (pseudocolored green) and mTFP (pseudocolored blue) reporter proteins shows four fibers. (Fii) 3D representation of PC space from the image in panel (Fi) shows similar behavior as the simulated image in panel (Ei) . (Fiii) Extraction of the cyan cluster (from black point to the white point via the inflection point) reconstructs the cyan fiber (Fiv) . (Fv) The same approach was applied to four other identifiable clusters of pixels and enough of the voxels from each cluster were extracted for all four of the fibers from the optic nerve in (Fi) .

Article Snippet: Tissue was subsequently incubated with primary antibodies against eYFP (chicken anti-GFP; 1:500; Kerafast), mCherry (rabbit anti-mCherry; 1:500; Kerafast), tagBFP (guinea pig anti-TFP; 1:500; Kerafast) and mTFP (rat anti-TFP; 1:500; Kerafast) for 3 days at 4°C.

Techniques: Injection, Immunohistochemical staining, Labeling, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Quantitative characterisation of ipRGCs in retinal degeneration using a computation platform for extracting and reconstructing single neurons in 3D from a multi-colour labeled population

doi: 10.3389/fncel.2022.1009321

Figure Lengend Snippet: Principal component analysis and spatial reconstruction of bipolar cells in the GRM6 Cre/+ retina. (A) Confocal tile-scanned image of a representative wholemount retina from an Grm6Cre/ +; mouse unilaterally intravitreally injected with the AAV Brainbow virus, following immunohistochemical labeling for mCherry (pseudocolored red), eYFP (pseudocolored green) and mTFP (pseudocolored blue) reporter proteins in en face (top) and Z (bottom) views. (B) The ROI from panel (A) following pre-filtering. (C) Spatial reconstruction of voxels from the four isolated clusters (Ci–iv) in an en face (top) and Z (bottom) views. (D) Filament tracer reconstructions of the four clusters isolated using the Brainbow analysis of individual neurons (BRIAN) platform (Di–iv) in an en face (top) and Z (bottom) views. (E) Spatial reconstruction of voxels from the four isolated clusters in an en face (top) and Z (bottom) views. (F) Filament tracer reconstructions of all four clusters isolated using the BRIAN platform an en face (top) and Z (bottom) views revealed 67 single cells, out of 76 in the starting image (A) . (G) Close up of a representative rod bipolar cell. Spatial reconstruction of voxels from one rod bipolar cell in Z (Gi) and XY (Giii) views. Filament tracer reconstruction of the rod bipolar cell in Z (Gii) and XY (Giv) views. (H) Close up of a representative cone bipolar cell. Spatial reconstruction of voxels from one cone bipolar cell in Z (Hi) and XY (Hiii) views. Filament tracer reconstruction of the cone bipolar cell in Z (Hii) and XY (Hiv) views.

Article Snippet: Tissue was subsequently incubated with primary antibodies against eYFP (chicken anti-GFP; 1:500; Kerafast), mCherry (rabbit anti-mCherry; 1:500; Kerafast), tagBFP (guinea pig anti-TFP; 1:500; Kerafast) and mTFP (rat anti-TFP; 1:500; Kerafast) for 3 days at 4°C.

Techniques: Injection, Immunohistochemical staining, Labeling, Isolation

Journal: Frontiers in Cellular Neuroscience

Article Title: Quantitative characterisation of ipRGCs in retinal degeneration using a computation platform for extracting and reconstructing single neurons in 3D from a multi-colour labeled population

doi: 10.3389/fncel.2022.1009321

Figure Lengend Snippet: Identifying ipRGC subtypes in the degenerate retina through quantitative morphological characteristics obtained using BRIAN. (A) Maximum projection of a representative wholemount retina from an Opn4 Cre/+ ;rd/rd mouse unilaterally intravitreally injected with the AAV Brainbow virus, following immunohistochemical labeling for mCherry (pseudocolored red), eYFP (pseudocolored green) and mTFP (pseudocolored blue) reporter proteins. (B) Representative filtered ROI from the boxed region in panel (A) shows dense labeling of Retinal Ganglion cells with considerable variation in immunofluorescence for the three reporter genes. (C) 3D representation of PC space (PC1 vs. PC2 vs. PC3) for all the voxels from the filtered ROI shown in panel (B) from which distinct clusters can be observed diverging from the “black point.” (D) En face views of the spatial distribution of voxels assigned to each of three distinct clusters in PC space from the ROI in panel (B) reveal the shape of 3 distinct cells. Color of voxels in each cluster (orange, green and pink) matches their appearance in the pseudocolored image in panel (B) . (E) Filament tracer reconstructions of 4 single ipRGCs isolated using the BRIAN platform from Opn4 Cre/+ ;rd/rd retinas in en face (top) and Z (bottom) views representative of M1 (Ei) , M1d (Eii) , M3 (Eiii) , and M6 (Eiv) subtypes. Note location of dendrites in outer portion of IPL (close to INL) for M1 cells and in both upper and lower IPL for M3 and M6; and soma displaced in the INL for the M1d (arrows denote axons). (F) Soma size and dendritic field diameter showed substantial variation across ipRGCs isolated using this method and stratifying exclusively in the inner sublamina of the IPL (putative M2, M4, and M5 cells appear as green, orange, and red symbols respectively); as did dendritic complexity as determined by Sholl analysis ( G ; color code as for panel F ). (H) Filament tracer reconstructions in both an en face (upper) and Z (lower) view of 3 ipRGCs isolated using the BRIAN platform representative of M5 (Hi) , M2 (Hii) , and M4 (Hiii) subtypes. Locations of inner nuclear layer (INL) and ganglion cell layer (GCL) provided as references for Z-projection images.

Article Snippet: Tissue was subsequently incubated with primary antibodies against eYFP (chicken anti-GFP; 1:500; Kerafast), mCherry (rabbit anti-mCherry; 1:500; Kerafast), tagBFP (guinea pig anti-TFP; 1:500; Kerafast) and mTFP (rat anti-TFP; 1:500; Kerafast) for 3 days at 4°C.

Techniques: Injection, Immunohistochemical staining, Labeling, Immunofluorescence, Isolation

Journal: Frontiers in Cellular Neuroscience

Article Title: Quantitative characterisation of ipRGCs in retinal degeneration using a computation platform for extracting and reconstructing single neurons in 3D from a multi-colour labeled population

doi: 10.3389/fncel.2022.1009321

Figure Lengend Snippet: Using BRIAN to trace axonal projections of ipRGCs in the Lateral Geniculate Nucleus (LGN). (A) Coronal Sections from an Opn4 Cre/+ ;rd/rd mouse brain following intravitreal AAV Brainbow injection. Immunohistochemical labeling for the mCherry reporter protein in monochrome reveals strong projections to regions including the SCN (suprachiasmatic Nucleus), OPN (Olivary Pretectal Nucleus), the dorsal and ventral LGN (Lateral Geniculate Nucleus) and the SC (Superior Colliculus). Schematic diagrams from the mouse atlas illustrate the densely stained regions in blue. (B) (Top) Confocal tile-scanned image of a 100 μm thick sagittal section of the Opn4 Cre/+ ;rd/rd dLGN following a unilateral intravitreal AAV Brainbow injection and immunohistochemical staining for the mCherry (pseudocolored red), eYFP (pseudocolored green) and mTFP (pseudocolored blue) reporter proteins. Inset: schematic diagram of a sagittal cross-section from the mouse atlas (above) illustrates anatomical position of the dLGN in blue. ROI from the boxed region following pre-filtering with BRIAN. (C) 3D representation of the PC space (PC1 vs. PC2 vs. PC3) for the filtered ROI shown in panel (B) which depicts all voxels in the filtered image. (D) 3 × 2D representations of the PCA space (from left to right; PC1 vs. PC2, PC1 vs. PC3 and PC2 vs. PC3) and the extraction polygon used to isolate the turquoise cluster. (E) Spatial reconstruction of voxels from the isolated turquoise cluster in panel (D) in both an en face (upper) and Z (lower) view. (F) Filament tracer reconstructions in both an en face (upper) and Z (lower) view of the turquoise cell isolated using the BRIAN platform. (G) Filament tracer reconstructions for the three single cell axonal projections (turquoise, pink and purple) combined in (top) XY and (bottom) XZ dimensions .

Article Snippet: Tissue was subsequently incubated with primary antibodies against eYFP (chicken anti-GFP; 1:500; Kerafast), mCherry (rabbit anti-mCherry; 1:500; Kerafast), tagBFP (guinea pig anti-TFP; 1:500; Kerafast) and mTFP (rat anti-TFP; 1:500; Kerafast) for 3 days at 4°C.

Techniques: Injection, Immunohistochemical staining, Labeling, Staining, Isolation

Journal: Frontiers in Cellular Neuroscience

Article Title: Quantitative characterisation of ipRGCs in retinal degeneration using a computation platform for extracting and reconstructing single neurons in 3D from a multi-colour labeled population

doi: 10.3389/fncel.2022.1009321

Figure Lengend Snippet: Using BRIAN to trace axonal projections of ipRGCs in the Suprachiasmatic Nucleus (SCN). (A) Confocal tile-scanned image of a representative of a 60 μm thick coronal section of the Opn4 Cre/+ ;rd/rd SCN (Suprachiasmatic Nucleus), following a unilateral intravitreal AAV Brainbow injection and immunohistochemical staining for the mCherry (pseudocolored red), eYFP (pseudocolored green) and mTFP (pseudocolored blue) reporter proteins following pre-filtering. Inset: schematic diagram of a coronal cross-section from the mouse atlas (above) illustrates anatomical position of the SCN in blue. (B) 3D representation of the PC space (PC1 vs. PC2 vs. PC3) for the filtered ROI shown in panel (A) which depicts all voxels in the filtered image. (C) Spatial reconstruction of voxels from the isolated red cluster in an en face view. (D) Filament tracer reconstructions in an en face view of the red cluster isolated using the BRIAN platform revealed 3 continuous filaments (which may represent the axonal projections of 3 cells with similar color or parts of the same neuron that would be continuous if viewed in a larger brain volume). (E) Spatial reconstruction of voxels from the isolated white cluster in an en face view. (F) Filament tracer reconstructions in an en face view of the white cluster isolated using the BRIAN platform. (G) Spatial location of voxels in en face view from 7 separate clusters (color of each voxel matches that of its appearance in the pseudocolored representation of this ROI in A ) isolated from the PCA (B) . (H) Filament tracer image of the seven voxel clusters in panel (G) in en face projection .

Article Snippet: Tissue was subsequently incubated with primary antibodies against eYFP (chicken anti-GFP; 1:500; Kerafast), mCherry (rabbit anti-mCherry; 1:500; Kerafast), tagBFP (guinea pig anti-TFP; 1:500; Kerafast) and mTFP (rat anti-TFP; 1:500; Kerafast) for 3 days at 4°C.

Techniques: Injection, Immunohistochemical staining, Staining, Isolation

Journal: eLife

Article Title: Inhibition of the Notch signal transducer CSL by Pkc53E-mediated phosphorylation to fend off parasitic immune challenge in Drosophila

doi: 10.7554/eLife.89582

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-mCherry , GeneTex , RRID: AB_2721247 ; Cat# GTX128508 , WB(1:1000).

Techniques: Recombinant, In Vitro, Expressing, Activation Assay, Control, Transfection, Construct, Activity Assay, Clone Assay, Knock-In, Mutagenesis, Knock-Out, Kinase Assay, Purification, Reporter Assay, Software, Sequencing

Journal: Oncogene

Article Title: Autophagy suppresses Ras-driven epithelial tumourigenesis by limiting the accumulation of reactive oxygen species

doi: 10.1038/onc.2017.175

Figure Lengend Snippet: Ras V12 expression induces autophagy. ( a – d ) Effect of Atg8a RNAi , Ras V12 and Ras V12 Atg8a RNAi expressed via the dpp-GAL4 driver on pmCherry-Atg8a expression in L3 wing discs. mCherry-Atg8a levels ( b ) are increased upon Ras activation. ( c ) mCherry-Atg8a punctae are detected in the Dpp domain (dotted lines) upon expression of Tsc1 and Tsc2 transgenes (positive control) or Ras V12 , while no puncta is detected upon expression of a control lacZ . ( d ) Non-cell-autonomous activation of autophagy is also observed in wild-type tissue surrounding Ras V12 and Ras V12 Atg8a RNAi tissue (arrowheads). ( e ) Monitoring of autophagy flux induction by detection of free mCherry in mCherry-Atg8a tissues. A 27 kDa band corresponding to free mCherry is detected in wing discs expressing Ras V12 and Ras V12 Atg8a RNAi in the Dpp domain, as well as in the positive control expressing Tor TED . *, unspecified band. ( f ) Effect of Atg8a RNAi , Ras V12 and Ras V12 Atg8a RNAi expressed via the dpp-GAL4 driver on GFP-Ref(2)P accumulation in L3 wing discs. Atg8a knockdown in the Dpp domain blocks autophagic flux as seen by accumulation of GFP-Ref(2)P aggregates. Slight accumulation of Ref(2)P aggregates is detected upon Ras activation, and blocking autophagic flux in this context leads to massive accumulation of Ref(2)P aggregates in the Dpp domain, quantified in ( g ). ( h ) As in the developing eye epithelium, autophagy inhibition in a Ras-activated background leads to tissue overgrowth, with proportion of GFP+ tissue higher in Ras V12 Atga- RNAi compared with Ras-only or Atg8a -RNAi-only controls. Scale bars: ( a and e ) 100 μm, ( c ) 20 μm and ( d ) 50 μm. Error bars: s.e.m. Statistics: one-way ANOVA with Tukey's multiple correction.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal anti-ref(2)p (1:10 000 (WB), 1:2000 (IF); a gift from G Juhász), rabbit polyclonal anti-Atg8a (1:5000 (WB); a gift from T Neufeld), mouse anti-MMP1 (1:100 (WB); Developmental Studies Hybridoma Bank, University of Iowa, IA, USA), mouse monoclonal anti-Elav (1:200; Developmental Studies Hybridoma Bank), rabbit polyclonal anti-β-galactosidase (1:200; Molecular Probes, Eugene, OR, USA), rabbit polyclonal anti-Dcp1 (1:200; Cell Signaling, Danvers, MA, USA; a kind gift of L Cheng), mouse monoclonal anti-activated ERK1/2 (phospho-ERK 1:5000; Sigma, St Louis, MO, USA), rabbit polyclonal anti-ERK1/2 (total-ERK 1:5000; Cell Signaling), mouse monoclonal anti-α-tubulin (1:10 000; Sigma), rabbit polyclonal anti-mCherry (1:6000; Kerafast, Boston, MA, USA; EMU109).

Techniques: Expressing, Activation Assay, Positive Control, Blocking Assay, Inhibition