Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: TRPV4 activation enhances the expression of GFAP. A Immunofluorescence images show GFAP protein expression profiles in rat retinal vertical slices acquired from sham-operated retinas (saline-injected; control) ( A1 ), 1 µM GSK101-injected retinas ( A2 ), and 10 µM GSK101-injected retinas ( A3 ). Retinas that received no GFAP antibody served as negative controls ( A4 ). Double immunofluorescence staining showing GFAP expression when the GFAP antibody was pre-adsorbed with its blocking peptide (BP) ( A5 ). Scale bar: 20 µm. B Representative immunoblots showing changes in GFAP protein levels in control and 10 µM GSK101-injected retinas. C Bar chart summarizing mean expression levels of GFAP under different conditions. n = 6. * p < 0.05 vs control

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Activation Assay, Expressing, Immunofluorescence, Injection, Double Immunofluorescence Staining, Blocking Assay, Western Blot

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: TRPV4 activation enhances TNF-α production in cultured Müller cells. A Morphology of cultured Müller cells. Scale bar: 50 µm. B GSK101 treatment enhanced GFAP protein levels in cultured Müller cells. n = 4. ** p < 0.01 and *** p < 0.001. C Cumulative changes in GFAP protein levels in control and GSK101 treatment groups. D – F Cumulative changes in mRNA levels of TNF-α ( D ), IL-1β ( E ), and IL-6 ( F ) in Müller cell extracts obtained after saline treatment (control) or GSK101 treatment for 24 h. Relative abundances of mRNA were quantified using the 2 −ΔΔct calculation method and are expressed as fold changes. n = 3 for all groups. * p < 0.05 vs control. G , GSK101 treatment led to enhancement of TNF-α protein level. H Cumulative changes in TNF-α protein levels in control and GSK101 treatment groups. n = 6. * p < 0.05 and *** p < 0.001

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Activation Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: TRPA1-Mediated Src Family Kinases Activity Facilitates Cortical Spreading Depression Susceptibility and Trigeminovascular System Sensitization

doi: 10.3390/ijms222212273

Figure Lengend Snippet: Co-localization of TRPA1 and SFKs, and effects of pYEEI, the SFKs activator, on the anti-TRPA1 antibody-reduced cortical susceptibility to CSD in the mouse brain slice. ( A , B ) Representative images of expression of SFKs in neurons and astrocytes of mouse cerebral cortices. ( C ) Representative images of co-localization of TRPA1 and SFKs in mouse cerebral cortices. Staining with DAPI indicated nucleus as shown in blue; staining with the anti-TRPA1 antibody was shown in red; staining with the anti-NeuN antibody indicated neurons and staining with the anti-GFAP antibody indicated astrocytes as shown in green. Double immune-labeling showed expression of SFKs in neurons and astrocytes of mouse cerebral cortices or co-expression of TRPA1 and SFKs in mouse cerebral cortices (white arrows, n = 3 per group). ( D ) Representative images of the coronal slice of mouse brain before (upper) and after (lower) CSD induced by ejection of 260 mM KCl in the cerebral cortical region. The same area of interest (AOI) along CSD wave front (pointed by the short arrow) was selected and used for all images. ( E ) The averaged gray level within the AOI was plotted against time to generate the CSD curve. ( F , G ) Effects of 0.015 µM anti-TRPA1 antibody (Anti-TRPA1) ( n = 7), 0.015 µM anti-TRPA1 antibody + 0.1 µM pYEEI ( n = 7), or 0.015 µM anti-TRPA1 antibody + 0.1 µM YEEI ( n = 8) on CSD latency (seconds, s) and CSD propagation rate (mm/minute, mm/min) in mouse brain slices. Group data were presented as mean ± SEM. Two-tailed unpaired t -test was used for comparison between pYEEI and YEEI group in the presence of anti-TRPA1 antibody. Significance differences were shown as * p < 0.05, ** p < 0.01.

Article Snippet: Slices were incubated in 10% goat serum (AR0009 Boster Biological Technology, Pleasanton, CA, USA) at room temperature (RT) for 2 h. Brain slices were incubated in anti-SFKs antibody (1:80, AF3389 R&D Systems, Minneapolis, MN, USA) with anti-NeuN antibody (1:500, MAB377 Merck, St. Louis, MO, USA), or anti-GFAP antibody (1:100, 3670 CST, Beverly, MA, USA) or anti-TRPA1 antibody (1:100, ACC-037 Alomone Labs, Jerusalem, Israel), respectively at 4 °C overnight.

Techniques: Slice Preparation, Expressing, Staining, Labeling, Two Tailed Test

Journal: PLoS ONE

Article Title: Hypoxia-induced inflammation: Profiling the first 24-hour posthypoxic plasma and central nervous system changes

doi: 10.1371/journal.pone.0246681

Figure Lengend Snippet: (a) AQP-4 immunostaining of retina in control (a), 1h (b) and 18h (c) animals. (d-f) Kir4.1 immunostaining. (g-i) GFAP immunostaining (green) and DAPI nuclei labeling (blue). (j-l) TUNEL (red, arrows) and DAPI (blue). (m, n) Bar graph of quantification of AQP-4 (m) and Kir4.1 (n) expression levels in the retina and correlation with retinal thickness. (o) Bar graph of quantification of GFAP + processes across the IPL and correlation with retinal thickness. (p) Bar graph of quantification of the number of TUNEL + cells in each retina layer per retinal section. GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; OPL: outer plexiform layer, ONL: outer nuclear layer.

Article Snippet: Retinae were immunostained with primary antibodies to detect and AQP-4 (1:50, mouse; catalog number sc-32739, Santa Cruz Biotechnology, Inc. Dallas, Texas, USA), Kir4.1 (1:200, rabbit, catalog number APC-035, Alomone Labs, Jerusalem, Israel) and glial fibrillary acidic protein (GFAP) (1:1000, rabbit; catalog number ab7260; Abcam, Cambridge, MA, USA).

Techniques: Immunostaining, Labeling, TUNEL Assay, Expressing

Journal: Neurobiology of disease

Article Title: TAF1 -gene editing alters the morphology and function of the cerebellum

doi: 10.1016/j.nbd.2019.104539

Figure Lengend Snippet: List of primary and secondary antibodies used in the study.

Article Snippet: Similar control experiments were performed for the other antibodies used in this study (data not shown). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibodies Anti- Abbreviation Host Company Catalog number Dilution a TATA-box binding protein factor 1 TAF1 Rabbit Sigma Aldrich AV32473 1:500 Calbindin-D28k Calbindin Rabbit Sigma Aldrich C2724 1:250 Low-Voltage-Activated CaV3.1 Calcium Channels CaV3.1 Rabbit Almone labs ACC-021 1:250 Glial fibrillary acidic protein GFAP Rabbit Dako Z0334 1:250 Ionizing calcium-binding adaptor molecule 1 Iba1 Rabbit Wako Labs 019-19741 1:250 Red fluorescent protein TagRFP tRFP Rabbit Envitrogen AB233 1:250 Tublin Tublin Mouse Promega G7121 1:250 Secondary antibodies Anti- Host Label Dilution Company Rabbit IgG Goat Alexa 488 1:250 Life technologies Rabbit IgG Goat Alexa 594 1:250 Promega Mouse IgG Goat Alexa 594 1:250 Promega Open in a separate window a Dilutions were from original stocks supplied by the vendors List of primary and secondary antibodies used in the study.

Techniques: Binding Assay

Journal: Neurobiology of disease

Article Title: TAF1 -gene editing alters the morphology and function of the cerebellum

doi: 10.1016/j.nbd.2019.104539

Figure Lengend Snippet: (A) Expression of calbindin was decreased in TAF1-edited animals as compared to naïve and CRISPR-control groups. Note also a decrease in the number of Calbindin positive Purkinje cells in TAF1-edited animals compared to control animals. B) Expression of GFAP (astrocytes marker) was decreased in TAF1-edited animals as compared to naïve and CRISPR-control groups. Note also a decrease in the number of GFAP positive cells in TAF1-edited animals compared to control animals. (C) Expression of Iba1 (microglia marker) was increased in TAF1-edited animals as compared to naïve and CRISPR-control groups. Note also increase in the number of Iba1 positive cells in TAF1-edited animals compared to control animals. (D) Summary of the number of Purkinje cells per linear density in each of the experimental conditions. Data are shown as mean ± S.E.M., n = 12 fields per animal, 4 animals per experimental condition. *p<0.05 versus; naïve, #p<0.05 versus gRNA-control (ANOVA followed by Tukey’s test). (E) and (F) Summary of the number of GFAP positive cells and Iba1 positive cells per linear density in each of the experimental conditions. Data are shown as mean ± S.E.M., n = 9 fields per animal, 3 animals per experimental condition. *p<0.05 versus; naïve, #p<0.05 versus gRNA-control (ANOVA followed by Tukey’s test). The experiments were conducted in an investigator blinded manner.

Article Snippet: Similar control experiments were performed for the other antibodies used in this study (data not shown). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibodies Anti- Abbreviation Host Company Catalog number Dilution a TATA-box binding protein factor 1 TAF1 Rabbit Sigma Aldrich AV32473 1:500 Calbindin-D28k Calbindin Rabbit Sigma Aldrich C2724 1:250 Low-Voltage-Activated CaV3.1 Calcium Channels CaV3.1 Rabbit Almone labs ACC-021 1:250 Glial fibrillary acidic protein GFAP Rabbit Dako Z0334 1:250 Ionizing calcium-binding adaptor molecule 1 Iba1 Rabbit Wako Labs 019-19741 1:250 Red fluorescent protein TagRFP tRFP Rabbit Envitrogen AB233 1:250 Tublin Tublin Mouse Promega G7121 1:250 Secondary antibodies Anti- Host Label Dilution Company Rabbit IgG Goat Alexa 488 1:250 Life technologies Rabbit IgG Goat Alexa 594 1:250 Promega Mouse IgG Goat Alexa 594 1:250 Promega Open in a separate window a Dilutions were from original stocks supplied by the vendors List of primary and secondary antibodies used in the study.

Techniques: Expressing, CRISPR, Marker