Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R expression in mouse retina. ( A ) Representative western blot and ( B ) protein quantification of P2X7R expression in C57BL/6J, rd10early, and rd10late mice. ( C ) Mean fluorescence values of P2X7R immunostaining in P2X7R-positive cells analyzed by flow cytometry in rd10early and rd10late mice compared with C57BL/6J mice. Student’s t -test * p < 0.05, ** p < 0.01. C57BL/6J1: postnatal day (P)20; C57BL/6J2: P40–P47; rd10early: P18 and rd10late: P44.

Article Snippet: The retinal cell suspension was then triple-stained with a cocktail of the following antibodies: rabbit anti-P2X7 receptor-ATTO633 (extracellular) (1:200; Alomone Labs), Rabbit anti-P2X4 receptor-FITC (extracellular) (1:200; Alomone Labs), and anti-CD11b-PE (Clone M/170; e-Bioscience, San Diego, CA, USA).

Techniques: Expressing, Western Blot, Fluorescence, Immunostaining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R expression in mouse retinas analyzed by immunohistochemistry. Cross-sectional cryosections of retinas from a representative C57BL/6J mouse ( A , D , G ) and rd10early ( B , E , H ) and rd10late ( C , F , I ) mice, stained with antibodies against P2X7R ( A – C , G – I ). Nuclei were stained with TO-PRO ( D – F ). In C57BL/6J mice, immunopositive fluorescence against P2X7R appears mainly located in the INL and GCL. RPE: retinal pigment epithelial cells; OS: outer segments; IS: inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar: 50 µm.

Article Snippet: The retinal cell suspension was then triple-stained with a cocktail of the following antibodies: rabbit anti-P2X7 receptor-ATTO633 (extracellular) (1:200; Alomone Labs), Rabbit anti-P2X4 receptor-FITC (extracellular) (1:200; Alomone Labs), and anti-CD11b-PE (Clone M/170; e-Bioscience, San Diego, CA, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X4R expression in mouse retina. ( A ) Representative western blot and ( B ) protein quantification of P2X4R expression in C57BL/6J, rd10early, and rd10late mice. ( C ) Mean fluorescence values of P2X4R immunostaining in P2X7R-positive cells analyzed by flow cytometry in rd10early and rd10late mice compared with the control expression in C57BL/6J mice. Student’s t -test, * p < 0.05. C57BL/6J1: postnatal day (P)20; C57BL/6J2: P40-P47; rd10early: P18 and rd10late: P44.

Article Snippet: The retinal cell suspension was then triple-stained with a cocktail of the following antibodies: rabbit anti-P2X7 receptor-ATTO633 (extracellular) (1:200; Alomone Labs), Rabbit anti-P2X4 receptor-FITC (extracellular) (1:200; Alomone Labs), and anti-CD11b-PE (Clone M/170; e-Bioscience, San Diego, CA, USA).

Techniques: Expressing, Western Blot, Fluorescence, Immunostaining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression in retinal myeloid cells (CD11b + ). The CD11b immunoreactive cell population was analyzed by flow cytometry. Bar graphs show the number of CD11b + cells expressing P2X7R ( A ) and the mean intensity of P2X7R fluorescence values ( B ). Also, the number of CD11b + cells expressing P2X4R is shown ( C ), and the mean intensity of P2X4R fluorescence values ( D ). C57BL/6J: postnatal day (P)47; rd10early: P18 and rd10late: P44. ** p < 0.01, *** p < 0.001.

Article Snippet: The retinal cell suspension was then triple-stained with a cocktail of the following antibodies: rabbit anti-P2X7 receptor-ATTO633 (extracellular) (1:200; Alomone Labs), Rabbit anti-P2X4 receptor-FITC (extracellular) (1:200; Alomone Labs), and anti-CD11b-PE (Clone M/170; e-Bioscience, San Diego, CA, USA).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression in the CD11b + population, analyzed by flow cytometry. Whole retinal cells from C57BL/6J, rd10early, and rd10late mice were triple-stained with antibodies against CD11b, P2X7R, and P2X4R. After discarding the cell debris and selecting singlets, CD11b + cells were gated ( A ) and the expression of P2X7R and P2X4R was analyzed ( B – D ). ( B ) Double contour plots representing P2X7R and P2X4R expression, showing P2X7R- and P2X4R-positive populations (green line) and P2X7R- and P2X4R-highly immunoreactive populations (blue line). Each plot shows the sum of a minimum of 3 independent replicates. ( C , D ) Bar graphs showing the number of double-positive cells (P2X7R and P2X4R) and mean fluorescence values for P2X7R and P2X4R in the whole double-positive population ( C ) and the highly immunoreactive double-positive population ( D ). Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001, ns: non-statistically significant. C57BL/6J: postnatal day (P)47; rd10early: P18 and rd10late: P44.

Article Snippet: The retinal cell suspension was then triple-stained with a cocktail of the following antibodies: rabbit anti-P2X7 receptor-ATTO633 (extracellular) (1:200; Alomone Labs), Rabbit anti-P2X4 receptor-FITC (extracellular) (1:200; Alomone Labs), and anti-CD11b-PE (Clone M/170; e-Bioscience, San Diego, CA, USA).

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression analysis in CD11b+ cells, assessed by flow cytometry. Whole retinal cells from C57BL/6J, rd10early, and rd10late mice were triple-stained with antibodies against CD11b, P2X7R, and P2X4R. After discarding the cell debris and selecting singlets, CD11b-immunopositive cells were gated and two populations were selected according to medium or high immunoreactivity against the CD11b antibody, as shown in the contour plot (rd10 postnatal day [P]18) ( A ). The dot plot shows the sum of a minimum of 3 independent replicates. The immunoreactivity against P2X7R ( B ) and P2X4R ( C ) was compared in the two CD11b populations of each group of mice. Student’s t -test was performed between the two populations in each condition. Data are presented as mean values ± SD. Student’s t -test, ** p < 0.01. C57BL/6J: P47; rd10early: P18 and rd10late: P44.

Article Snippet: The retinal cell suspension was then triple-stained with a cocktail of the following antibodies: rabbit anti-P2X7 receptor-ATTO633 (extracellular) (1:200; Alomone Labs), Rabbit anti-P2X4 receptor-FITC (extracellular) (1:200; Alomone Labs), and anti-CD11b-PE (Clone M/170; e-Bioscience, San Diego, CA, USA).

Techniques: Expressing, Flow Cytometry, Staining