Journal: bioRxiv

Article Title: Green spotted puffers can detect an almost nontoxic TTX analog odor using crypt olfactory sensory neurons

doi: 10.1101/2021.09.16.460554

Figure Lengend Snippet: A~D: pS6 immunohistochemistry of OE cross-sections exposed to Vehicle (ACSF; A), L-Arg (10 -6 M; B), 5,6,11-trideoxyTTX (10 -6 M; C), or TTX (10 -6 M; D). Arrow: activated OSNs. Scale bar: 10 μm. Insets in B and C: Magnified view of pS6-immunopositive OSNs. Arrowhead: Cilia of activated OSNs. Scale bar: 2.5 μm. E: Lower magnification confocal microscopy image of pS6-immunopositive OSNs in OE cross section exposed to 5,6,11-trideoxyTTX. Arrow: 5,6,11-trideoxyTTX-activated OSNs. Scale bar: 50 μm.

Article Snippet: For single immunohistochemistry, sections were incubated successively in PBST with 1) an anti-phosphorylated ribosomal protein S6 (pS6) rabbit polyclonal antibody (Ser244, Ser247 anti-pS6, 44-923G, Thermo Fisher Scientific, MA, USA; 1:5000) that was used in mouse OSNs as validated in as a neural activity marker for 2 days at 4°C; 2) an Alexa Fluor® 488-conjugated secondary antibody (goat anti-rabbit IgG (H+L), A-11008, Thermo Fisher Scientific, 1:500) for 1 day at 4°C; and 3) 4’,6-diamidino-2-phenylindole (DAPI; 1:1000 dilution) for 5 min at room temperature.

Techniques: Immunohistochemistry, Confocal Microscopy

Journal: bioRxiv

Article Title: Green spotted puffers can detect an almost nontoxic TTX analog odor using crypt olfactory sensory neurons

doi: 10.1101/2021.09.16.460554

Figure Lengend Snippet: A~C: Reconstructed confocal microscopy images of 5,6,11-trideoxyTTX-administered OE displaying pS6-immunopositive OSNs (A) or S100-immunopositive OSNs (B) or stained with DAPI (C). D: A merged image of A to C (pS6: magenta, S100: green, DAPI: blue). Each immunopositive OSN had apical invagination, and their cell bodies were located in the superficial layer of the OE. Arrow: Nucleus of 5,6,11-trideoxyTTX-activated OSNs. Scale bar: 10μm. E: No pS6-immunopositive OSN was observed in Vehicle (ACSF)-treated OE. Scale bar: 10μm.

Article Snippet: For single immunohistochemistry, sections were incubated successively in PBST with 1) an anti-phosphorylated ribosomal protein S6 (pS6) rabbit polyclonal antibody (Ser244, Ser247 anti-pS6, 44-923G, Thermo Fisher Scientific, MA, USA; 1:5000) that was used in mouse OSNs as validated in as a neural activity marker for 2 days at 4°C; 2) an Alexa Fluor® 488-conjugated secondary antibody (goat anti-rabbit IgG (H+L), A-11008, Thermo Fisher Scientific, 1:500) for 1 day at 4°C; and 3) 4’,6-diamidino-2-phenylindole (DAPI; 1:1000 dilution) for 5 min at room temperature.

Techniques: Confocal Microscopy, Staining

Journal: Translational Oncology

Article Title: Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer

doi: 10.1016/j.tranon.2020.100828

Figure Lengend Snippet: Combined CB-839/anti-EGFR mAb therapy overcomes intrinsic cetuximab-resistance in SW48 xenografts in vivo . (A) Mice with SW48 cell line xenografts, a model of intrinsic cetuximab resistance, were treated with either vehicle (black), CB-839 (red), cetuximab (blue) or cetuximab and CB-839 (purple) for 30 days and tumor volumes were monitored (n = 5 mice/group). (B) Representative images of mice treated with cetuximab alone (left) or cetuximab in combination with CB-839 (right). Mice progressed on single agent CB-839 or cetuximab yet exhibit reduced tumor volume with combination cetuximab and CB-839. (C) Immunohistochemistry (IHC) was performed on tumors from each of the treatment groups to evaluate Ki67 and pS6 expression. Shown are representative images (40×). The number of positive cells were counted in regions of interest on images from Ki67 IHC (D) and pS6 IHC (E) (n = 3–15 fields of view, at least 3 animals/treatment group). Reduced Ki67 and pS6 were observed when treated with CB-839 in combination with cetuximab compared to cetuximab or CB-839 alone. (F) Representative H&E images (0.46×) of tumor tissues from each of the treatment groups. (G) IHC was performed on tumors from each of the treatment groups to evaluate cleaved caspase-3 expression. Shown are representative images (20×). (H) The number of positive cells were counted in regions of interest on images from cleaved caspase-3 IHC (n = 3–15 fields of view, at least 3 animals/treatment group). Error bars represent ± SD. Statistical significance is defined as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The samples were incubated with pS6 Ribosomal Protein rabbit monoclonal antibody (Cell Signaling, #4858) at a dilution of 1:500 overnight.

Techniques: In Vivo, Immunohistochemistry, Expressing

Journal: Translational Oncology

Article Title: Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer

doi: 10.1016/j.tranon.2020.100828

Figure Lengend Snippet: Combined CB-839/anti-EGFR mAb therapy overcomes acquired cetuximab-resistance in HCA-7 CC-CR xenografts in vivo . (A) Mice with HCA-7 CC-CR cell line xenografts, a model of acquired cetuximab-resistance, were treated with either vehicle (black), CB-839 (red), cetuximab (blue) or cetuximab and CB-839 (purple) for 21 days. At this time point, treatment was stopped (dotted line). Tumor volumes were monitored to day 57 (n = 8 mice/group). Mice progressed on single agent CB-839 or cetuximab yet exhibit reduced tumor volume with combination cetuximab and CB-839. (B) IHC was performed on tumors from each of the treatment groups to evaluate Ki67 and pS6 expression. Shown are representative images (40×). The number of positive cells were counted in regions of interest on images from Ki67 IHC (C) and pS6 IHC (D) (n = 3–15 fields of view, at least 3 animals/treatment group). Reduced Ki67 and pS6 were observed when treated with CB-839 in combination with cetuximab compared to cetuximab or CB-839 alone. (E) Representative H&E images (0.46×) of tumor tissues from each of the treatment groups. (F) IHC was performed on tumors from each of the treatment groups to evaluate cleaved caspase-3 expression. Shown are representative images (20×). (G) The number of positive cells were counted in regions of interest on images from cleaved caspase-3 IHC (n = 3–15 fields of view, at least 3 animals/treatment group). Error bars represent ± SD. Statistical significance is defined as follows: # p = 0.0574, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The samples were incubated with pS6 Ribosomal Protein rabbit monoclonal antibody (Cell Signaling, #4858) at a dilution of 1:500 overnight.

Techniques: In Vivo, Expressing

Journal: Proceedings of the Royal Society B: Biological Sciences

Article Title: Behavioural and neural correlates of social hierarchy formation in a sex-changing fish

doi: 10.1098/rspb.2024.2097

Figure Lengend Snippet: Neural activation patterns after social disruption. Representative images for each brain area are depicted on the left (i; rank 2) and middle (ii; ranks 3−5) in each panel. Boxplots of pS6 counts are depicted on the right of each panel, with 2c and 3−5c representing the controls (iii). (A) The medial part of the dorsal telencephalon (Dm), with magnification of the insets to the right of each panel (scale bar of insets = 20 µm). (B) The dorsal part of the ventral telencephalon (Vd). (C) The ventral part of the ventral telencephalon (Vv). (D) The supracommissural nucleus of the ventral telencephalon (Vs). (E) The central nucleus of the telencephalic area (Vc). (F) the preoptic area (POA). (G) the ventral tuberal region of the hypothalamus (vTn). (H) the periaqueductal gray (PAG). (I) the periventricular nucleus of the posterior tuberculum (TPp). (J) The anterior tuberal nucleus (aTn; ). Scale bar = 100 µm. (K) Overview of brain regions with significantly different pS6 counts in rank 2 fish compared to other ranks.

Article Snippet: Non-specific binding was blocked (1% bovine serum albumin, 0.3% Triton-X and 5.0% normal goat serum made in 1 × PBS) for 2 h at room temperature, and slides were incubated with primary pS6 antibody (1 : 1500; Cell Signaling Technologies pS6 ribosomal protein S235/236 rabbit monoclonal antibody no. 4858) overnight at 4°C.

Techniques: Activation Assay, Disruption

Journal: The Journal of Comparative Neurology

Article Title: Peculiar protrusions along tanycyte processes face diverse neural and nonneural cell types in the hypothalamic parenchyma

doi: 10.1002/cne.24965

Figure Lengend Snippet: Primary antibodies used in the study

Article Snippet: The rabbit polyclonal antibody to pS6 (phospho‐S6 Ribosomal protein) (Cell Signaling Technology Cat# 2211, RRID:AB_331679) was validated by the manufacturer: it stains the expected band at 32 kDa on western blots.

Techniques: Recombinant, Sequencing, Derivative Assay, Purification, Isolation, Membrane, Phospho-proteomics

Journal: The Journal of Comparative Neurology

Article Title: Peculiar protrusions along tanycyte processes face diverse neural and nonneural cell types in the hypothalamic parenchyma

doi: 10.1002/cne.24965

Figure Lengend Snippet: Organelle composition of tanycyte protrusions. (a–c) High‐magnification z‐stack images (×63) showing the distribution of tdTomato (red, a), TOMM20 immunoreactivity (green, b) and merge (yellow, c) in coronal section in the arcuate nucleus, in Zone 2. (d–f) High‐magnification z‐stack images (×63) showing the distribution of tdTomato (red, d), phospho‐S6 immunoreactivity (pS6, green, e) and merge (yellow, f) in coronal section in the arcuate nucleus, in Zone 2. (g–i ) High‐magnification z‐stack images (×63) showing the distribution of tdTomato (red, g) and NPY‐GFP (green, g), pS6 immunoreactivity (blue, h) and merge (pink, i) in coronal section in the arcuate nucleus, in Zone 2. (j–l) High‐magnification z‐stack images (×63) showing the distribution of tdTomato (red, j) and POMC‐GFP (green, j), pS6 immunoreactivity (blue, k) and merge (pink, l) in coronal section in the arcuate nucleus, in Zone 2. Pictures (a–l) are single planes of z‐stack acquisition. Inset on the top in a–l panels shows the orthogonal view on the horizontal line; inset on the right in a–l panels shows the orthogonal view on the vertical line. Arrows and arrowheads indicate colocalization observed or not, respectively, in tanycytes. Empty arrows indicate colocalization observed in neurons. Low magnifications images are available upon request. Scale bars = 10 μm in a–l [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: The rabbit polyclonal antibody to pS6 (phospho‐S6 Ribosomal protein) (Cell Signaling Technology Cat# 2211, RRID:AB_331679) was validated by the manufacturer: it stains the expected band at 32 kDa on western blots.

Techniques: