Journal: Toxicology Reports

Article Title: Short-term whole body cigarette smoke exposure induces regional differences in cellular response in the mouse larynx

doi: 10.1016/j.toxrep.2021.04.007

Figure Lengend Snippet: Cleaved caspase-3 (CC3) apoptotic activity in VF. CSE did not alter CC3 labeled apoptotic cell populations after 1 day of CSE (B, F), 5 days of CSE (C, G), and 10 days of CSE (E, H) when compared to control (A). No alterations were seen in mice in the 5 day REV group (D, G). At day 1, n = 3 in control and CSE groups. At day 5, n = 4 in control and n = 5 in CSE groups. n = 5 in 5 day REV group. At day 10, n = 4 in control and n = 3 in the CSE group. In panel (A–E), purple channel images are hematoxylin stained nuclei and brown channel images are DAB substrates indicating CC3 positive nuclei. Red circles indicate CC3 labeled apoptotic cells. Images are at a magnification of 40x. Bar graphs show the mean with SD.

Article Snippet: To detect apoptosis, the slides were immunohistochemically stained with antibodies against cleaved caspase-3 (CC3) using the ImmPRESS Excel Amplified HRP Polymer kit for anti-Rabbit IgG’s (MP-7601, Vector laboratories, CA, USA).

Techniques: Activity Assay, Labeling, Staining

Journal: Toxicology Reports

Article Title: Short-term whole body cigarette smoke exposure induces regional differences in cellular response in the mouse larynx

doi: 10.1016/j.toxrep.2021.04.007

Figure Lengend Snippet: Cleaved caspase-3 (CC3) apoptotic activity in subglottis. Elevated numbers of CC3 positive cells were found only at day 1 after CSE (B, F) in comparison to the controls (A). 5 day CSE (C, G), 5 day REV (D, G), and 10 day CSE (E, H) groups did not have any significant increase in CC3 levels. At day 1, n = 3 in control and CSE groups. At day 5, n = 4 in control and CSE groups. n = 4 in the 5 day REV group. At day 10, n = 3 in control and CSE groups. In panel (A–E), purple channel images are hematoxylin stained nuclei and brown channel images are DAB substrate indicating CC3 positive nuclei. Red circles indicate CC3 labeled apoptotic cells. Images are at a magnification of 40x. Bar graphs show the mean with SD. * p ≤ 0.05.

Article Snippet: To detect apoptosis, the slides were immunohistochemically stained with antibodies against cleaved caspase-3 (CC3) using the ImmPRESS Excel Amplified HRP Polymer kit for anti-Rabbit IgG’s (MP-7601, Vector laboratories, CA, USA).

Techniques: Activity Assay, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: Complement Targets Newborn Retinal Ganglion Cells for Phagocytic Elimination by Microglia

doi: 10.1523/JNEUROSCI.1854-18.2018

Figure Lengend Snippet: Microglia are phagocytic at embryonic stages and interact with RGCs. A, High-resolution confocal image of microglial engulfment of an apoptotic cell (CC3+): DAPI (blue), Cx3cr1-gfp+ microglia (green), Brn3+ RGCs (red), and CC3+ (white). B, Microglia engulfing Brn3+ RGC. Right, bottom, Z planes: Cx3cr1-gfp+ microglia (green) and Brn3+ RGCs (red). C, D, Microglia interacting with non-CC3+ RGCs: DAPI (blue), Cx3cr1-gfp+ microglia (green), Brn3+ RGCs (red), and CC3+ (white). E–I, IMARIS-based 3D reconstruction of microglia interacting with RGCs: Cx3cr1-gfp+ microglia (green), Brn3+ RGCs (red), and CC3+ (white). F, Reconstruction of C. G, Reconstruction of D. J, Genes associated with phagocytosis assessed at e12.5 relative to P0 by whole-retina qRT-PCR. Normalized to β actin (n = 3 each; Mertk, t(4) = 18.74, p < 0.0001; Cd68, t(4) = 6.71, p = 0.0026; t(4) = 16.41; C1qb, p < 0.0001; C3, t(4) = 7.33, p = 0.0018). qPCR graph represents fold change. Error bar indicates the SEM of δ Ct values. K, Percentage of Brn3+ RGC density in depleted retinas normalized to littermate control retinas at e20.5/P0 (n = 5 retinas/3 animals each; one-sample t test, t(4) = 6.898, p = 0.0023). Scale bars: E, F, 10 μm; G, 3 μm; H, I, 5 μm. Unpaired Student's t test was used to determine significance of δ Ct values. ****p < 0.0001. **p < 0.01. Movie 4 is of C, F. Movie 3 is of D, G. Movie 1 is of H. Movie 2 is of I.

Article Snippet: Primary antibodies used are as follows: goat Brn3 (1:50, Santa Cruz Biotechnology sc-6026), rabbit phospho-histone H3 (pH3) (1:500, Millipore 06-570), rabbit cleaved caspase-3 (CC3, 1:500, BD Biosciences 559565), mouse Rxrγ (1:250, Santa Cruz Biotechnology sc-365252), mouse Brn3a (1:50, EMD Millipore MAB1585), rabbit calbindin (1:2000, EMD Millipore PC253L), goat GFP (1:2000, Abcam ab5450), rabbit Iba1 (1:1000, Wako 019-19741), mouse BrdU (1:500, Thermo Fisher Scientific {"type":"entrez-nucleotide","attrs":{"text":"B35128","term_id":"2534497","term_text":"B35128"}} B35128 ), mouse Cd68 (1:300,Bio-Rad MCA1957), rabbit C1q (11500, Abcam ab182451), and rat Cd11b (1:50, BD Biosciences 563890).

Techniques: Quantitative RT-PCR

Journal: The Journal of Neuroscience

Article Title: Complement Targets Newborn Retinal Ganglion Cells for Phagocytic Elimination by Microglia

doi: 10.1523/JNEUROSCI.1854-18.2018

Figure Lengend Snippet: Microglial density is dynamic in the developing retina, and microglia primarily associate with newborn neurons. A–C, Representative confocal images of retinal cross sections and central regions (A′–C′) at e12.5, e16.5, and P0: DAPI (blue), Iba1 (purple), Cx3cr1-gfp (green), and Brn3 (red). Dashed line indicates the boundary between the retina and vitreous where Iba1+GFP+ vitreal macrophages reside (yellow arrowheads). NbL and DCL outlined in B′. D, qRT-PCR of whole-retina samples over embryonic development. Levels of Cx3cr1 and Iba1 expression at various ages relative to P60 and normalized to β actin (n ≥ 3 each). qPCR graph represents fold change relative to P60. Error bar indicates the SEM of δ Ct values. E, Microglial density in DCL and NbL at e12.5, e14.5, and e16.5. [n = 4, 4, and 6 animals, respectively; two-way ANOVA: interaction, F(2,22) = 9.265, p = 0.0012; age, F(2,22) = 33.12, p < 0.0001; retinal layer, F(2,22) = 33.12, p < 0.0001; Sidak's multiple-comparisons: DCL vs NbL at e12.5, t(22) = 8.942, p < 0.0001; e14.5, t(22) = 3.206, p = 0.0122; e16.5, t(22) = 7.24, p < 0.0001; Sidak's multiple-comparisons comparing age: e12.5 vs e14.5, t(22) = 6.86, p < 0.0001; e12.5 vs e16.5, t(22) = 7.02, p < 0.0001; e14.5 vs e16.5, t(22) = 0.493, p = 0.948]. F, High-resolution confocal images with the Z plane of Iba1+ microglia in the NbL in contact with a migrating Brn3+ RGC (left) or not in contact (right). Iba1+ microglia (green) and Brn3+ RGCs (red). Z plane shown to the right and bottom of images. White cross represents point of interest. G, Percentage of Iba1+ microglia in the NbL-contacting Brn3+ RGCs at e12.5, e14.5, and e16.5 (n = 4, 4, and 6 animals, respectively). H, Percentage total Iba1+ microglia contacting CC3+ cells at e12.5 and e16.5 (n = 4 retinas, e12.5; n = 7 retinas, e16.5). Scale bars: 100 μm for whole-retina cross sections, 50 μm for high-magnification central region, and 10 μm for microglia contact in NbL. Graphs represent the mean; error bars indicate SEM. Four or five sections per retina were analyzed. ****p < 0.0001. *p < 0.05.

Article Snippet: Primary antibodies used are as follows: goat Brn3 (1:50, Santa Cruz Biotechnology sc-6026), rabbit phospho-histone H3 (pH3) (1:500, Millipore 06-570), rabbit cleaved caspase-3 (CC3, 1:500, BD Biosciences 559565), mouse Rxrγ (1:250, Santa Cruz Biotechnology sc-365252), mouse Brn3a (1:50, EMD Millipore MAB1585), rabbit calbindin (1:2000, EMD Millipore PC253L), goat GFP (1:2000, Abcam ab5450), rabbit Iba1 (1:1000, Wako 019-19741), mouse BrdU (1:500, Thermo Fisher Scientific {"type":"entrez-nucleotide","attrs":{"text":"B35128","term_id":"2534497","term_text":"B35128"}} B35128 ), mouse Cd68 (1:300,Bio-Rad MCA1957), rabbit C1q (11500, Abcam ab182451), and rat Cd11b (1:50, BD Biosciences 563890).

Techniques: Quantitative RT-PCR, Expressing

Journal: The Journal of Neuroscience

Article Title: Complement Targets Newborn Retinal Ganglion Cells for Phagocytic Elimination by Microglia

doi: 10.1523/JNEUROSCI.1854-18.2018

Figure Lengend Snippet: Inhibition of microglia does not change retinal cell apoptosis or alter microglial contact with apoptotic cells. A, Representative retinal cross sections of control and minocycline-treated animals at e16.5 with higher magnification (C′): DAPI (blue) and CC3+ apoptotic cells (white). B, CC3+ cells at e16.5 of control and minocycline-treated retinas [n = 6 animals each; two-way ANOVA: interaction, F(1,20) = 0.039, p = 0.846; treatment, F(1,20) = 0.597, p = 0.449; retinal layer, F(1,20) = 0.9473, p = 0.342; Sidak's multiple-comparisons: minocycline vs control DCL, t(20) = 0.685, p = 0.7511; NbL, t(20) = 0.407, p = 0.903]. C, Number of CC3+ cells at e16.5 contacted by microglia (n = 7 each; t(12) = 0.3712, p = 0.717). D, Average distance of microglia to a CC3+ cell. Each dot represents the mean distance for a particular retina (n = 7 each; t(12) = 1.162, p = 0.268). E, CC3+ cells at e13.5 of control and minocycline-treated retinas (n = 3 animals each; t(4) = 0.498, p = 0.645). Scale bars: 100 μm for whole-retina cross sections (A) and 50 μm higher-magnification central regions (A′). Graphs indicate mean with SEM. Individual dots represent individual retinas.

Article Snippet: Primary antibodies used are as follows: goat Brn3 (1:50, Santa Cruz Biotechnology sc-6026), rabbit phospho-histone H3 (pH3) (1:500, Millipore 06-570), rabbit cleaved caspase-3 (CC3, 1:500, BD Biosciences 559565), mouse Rxrγ (1:250, Santa Cruz Biotechnology sc-365252), mouse Brn3a (1:50, EMD Millipore MAB1585), rabbit calbindin (1:2000, EMD Millipore PC253L), goat GFP (1:2000, Abcam ab5450), rabbit Iba1 (1:1000, Wako 019-19741), mouse BrdU (1:500, Thermo Fisher Scientific {"type":"entrez-nucleotide","attrs":{"text":"B35128","term_id":"2534497","term_text":"B35128"}} B35128 ), mouse Cd68 (1:300,Bio-Rad MCA1957), rabbit C1q (11500, Abcam ab182451), and rat Cd11b (1:50, BD Biosciences 563890).

Techniques: Inhibition

Journal: Frontiers in Immunology

Article Title: Dynamics of necroptosis in kidney ischemia-reperfusion injury

doi: 10.3389/fimmu.2023.1251452

Figure Lengend Snippet: Comparison of cleaved Caspase-3 staining in WT and Mlkl -ko mice at 24 hrs post-reperfusion. (A) : WT; (B) : Mlkl -ko; (C) : sham; (D) : semi-quantitative analysis. Mann Whitney U test with * p<0.05 compared to sham procedure and # p<0.05 compared to WT mice. Data presented as mean ± SEM. Image magnification x 200.

Article Snippet: Sections were then incubated with rabbit monoclonal anti-phospho-MLKL (pSer345) (ab196436, 1:100, Abcam, Melbourne, Australia) or rabbit monoclonal anti-human/mouse cleaved Caspase-3 (cC3) (Asp175) (IC835G, 1:200, RD Systems, Noble Park, Australia) in 2% swine serum at 4°C overnight.

Techniques: Comparison, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD11c-expressing microglia are transient, driven by interactions with apoptotic cells

doi: 10.1101/2024.06.24.600082

Figure Lengend Snippet: (A) Confocal image of P3 retinal flat mount (Arrowhead and magnified boxes illustrate interactions) in NFL/GCL. CD11c-GFP+ microglia (green); cleaved caspase 3, CC3 (pink); and RBPMS (blue). Scale bar, 50μm. (B) Percent CD11c-GFP+ microglia contacting apoptotic RGCs (RBPMS+CC3+) at P3 out of total CD11c-GFP+ microglia within GCL. (n=5 animals). (C) Confocal images of retinal flat mounts of P3 CD11c-GFP animals in GCL showing a range of microglial GFP expression from none to high. Scale bar, 10μm. Dashed line demarcates distinction between CD11c-GFP negative and positive. (D) Cell area immunostained for CD68 ( Top ) and C1q ( Bottom ) in CD11c-GFP- and CD11c-GFP+ microglia (n=4 animals, 352 GFP-cells and 422 GFP+ cells). Mann-Whitney test Top **P =. 0011 and Bottom ****P <. 0001. (E) Scatter plot of CD68 area/cell compared to CD11c-GFP area/cell at P3 in GCL. Pearson r= 0.4145 **** P < .0001. (F) Flow cytometry analysis showing the percent CD11c Hi of total CD45+CD11b+ or CD45+CX3CR1-GFP+ microglia from retinas across all genotypes. CX3CR1-GFP/+ (n=10), CX3CR1-KO (n=6), CR3 KO (n=6), MerTK KO (n=9), Axl KO (n=8), and MerTK/Axl dKO (n=6), ≥ 2 litters collected for each genotype, ± SEM. One-way ANOVA F(5, 39) = 34.64 P <.0001 and Tukey’s multiple comparisons. **** P <.0001, *** P =.0003, ** P =.0056, * P =.02, NS, not significant. (G) Max projected confocal image of P5 retinal flat mount from WT and AXL KO, Spp1+ (green) and total microglia (IBA1+; red). Scale bar, 100μm. (H) Density of Spp1+IBA1+ microglia in AXL WT and KO (n=5, n=4 animals) ± SEM. Unpaired t test * P = .0139. (I) Percent Spp1+IBA1+ of total IBA1+ microglia in AXL WT and KO (n=5, n=4 animals) ± SEM. Unpaired t test ***P <.001

Article Snippet: Antibodies used include Goat polyclonal anti-GFP ( :2000, Abcam ab5450, RRID: AB_304897), Rabbit monoclonal anti-C1q ( :1500, Abcam an182451, RRID: AB_2732849), Rabbit polyclonal anti-IBA1 ( :1000, Wako 019-19741, RRID: AB_839504), Rabbit monoclonal anti-CC3 ( :500, BD Biosciences 559565, RRID: AB_397274), Guinea pig polyclonal anti-RBPMS ( :750, Millipore Sigma ABN1376, RRID: AB_2687403), Rat monoclonal anti-CD68 ( :250, Bio-Rad MCA1957, RRID: AB_322219), and Goat polyclonal anti-Osteopontin ( :100, Thermo Fischer PA5-34579, RRID: AB_2551931).

Techniques: Expressing, MANN-WHITNEY, Flow Cytometry

Journal: bioRxiv

Article Title: CD11c-expressing microglia are transient, driven by interactions with apoptotic cells

doi: 10.1101/2024.06.24.600082

Figure Lengend Snippet: (A) Depiction of two depletion strategies, Diphtheria toxin (DT) targeted ablation of CD11c-DTR/GFP+ microglia (Top) and PLX3397 (PLX)-mediated depletion to target more homeostatic microglia (Bottom). (B) Confocal images of CD11c-DTR/GFP+ microglia (green) in retinal flat mounts at P5 of Vehicle and DT-treated CD11c-DTR/GFP mice. Scale bar, 50μm. (C) Density of CD11c-DTR/GFP+ microglia in vehicle and DT-treated CD11c-DTR/GFP (n=6, n=7 animals) ± SEM. Unpaired t test **** P <.0001. (D) Confocal images of C1q+ microglia (white) in immunostained retinal flat mounts at P5 of vehicle and DT-treated CD11c-DTR/GFP mice. Scale bar, 50μm. (E) Densities of C1q+ cells in retinas of vehicle and DT-treated CD11c-DTR/GFP mice (n=6, n=7 animals) ± SEM. Mann Whitney test *P = .0140. (F) Max projected confocal images of C1q+ microglia in retinal flat mounts at P5 of vehicle and PLX-treated Cx3CR1/GFP+ mice. C1q (white). Scale bar, 50μm. (G) Density of C1q+ cells in retinas of naïve and PLX-treated CX3CR1-GFP (n=6, n=8) ± SEM. Unpaired t test *P = .0239. (H) Max projected confocal images of apoptotic RGCs in retinal flat mounts at P5 of all conditions/genotypes. CC3 (magenta); RBPMS (green). Scale bar, 50μm. (I) Density of CC3+RBPMS+ cells in retinas from vehicle and DT-treated CD11c-DTR/GFP mice and DT-treated wildtype mice (WT) (n=8, n=10, n=10 animals respectively) ± SEM. Ordinary one-way ANOVA **P = .0018 and Tukey’s multiple comparisons test: Veh vs DT ** P =.006, WT+DT vs DT **P = .0043, NS , not significant. (J) Density of CC3+RBPMS+ cells in naïve and PLX-treated CX3CR1-GFP/+ (n=8, n=8 animals) ± SEM. Unpaired t test **** P <.0001 (K) Scatter plot of dying RGC density (CC3+RBPMS+/mm 2 ) compared to microglial density (C1q+/mm 2 ) of same animal (n=4 CD11c + vehicle, n=5 CD11c + DT, n=10 PLX animals). Pearson r= -0.6350 ** P =.0035.

Article Snippet: Antibodies used include Goat polyclonal anti-GFP ( :2000, Abcam ab5450, RRID: AB_304897), Rabbit monoclonal anti-C1q ( :1500, Abcam an182451, RRID: AB_2732849), Rabbit polyclonal anti-IBA1 ( :1000, Wako 019-19741, RRID: AB_839504), Rabbit monoclonal anti-CC3 ( :500, BD Biosciences 559565, RRID: AB_397274), Guinea pig polyclonal anti-RBPMS ( :750, Millipore Sigma ABN1376, RRID: AB_2687403), Rat monoclonal anti-CD68 ( :250, Bio-Rad MCA1957, RRID: AB_322219), and Goat polyclonal anti-Osteopontin ( :100, Thermo Fischer PA5-34579, RRID: AB_2551931).

Techniques: MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Role of GRK2 in Trophoblast Necroptosis and Spiral Artery Remodeling: Implications for Preeclampsia Pathogenesis

doi: 10.3389/fcell.2021.694261

Figure Lengend Snippet: Suppression of GRK2 in placenta compromises placentation via necroptosis. (A) The 1st panel: Representative H&E staining images of E18.5 placentae from NC-LV and GRK2-LV groups. GRK2-LV placentae display a lack of vascularization, smaller placental size and a smaller labyrinth. Scale bar, 500 μm. Yellow lines, labyrinth and junction zone boundary; Green lines, junction zone and decidua boundary. The 2nd panel: Higher magnification of the upper white rectangle. Scale bar, 100 μm. 2nd and 3rd panels: The H&E staining and Von Kossa staining illustrate placental infarction and calcium deposition (red arrows) in GRK2-knockdown mice, respectively. (B) Volcano plots show differential expression for comparisons between GRK2-LV and NC-LV placentae ( N = 3 per group. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for the differentially expressed genes in total RNA-seq: GRK2-LV vs. NC-LV groups ( N = 3 per group). (D) TEM images show extensive necrosis in labyrinth layer of GRK2-LV group. Yellow dotted line, cell membranes; Yellow arrows swelling mitochondria. From upper to bottom, 500×, 1,500×, 2,500× magnification, respectively. (E,F) Western blot analysis of placental cleaved-caspase 3, RIPK1, RIPK 3, phospho-MLKL (p-MLKL; S358), and β-actin (loading control). The DNA fragment analysis (G,H) and apoptosis rate analysis (I) in mice term placentae. Green signal indicates TUNEL-positive nuclei. Scale bar, 50 μm. (J,K) The DNA fragment in patients’ placentae. Scale bar, 100 μm. Dec, decidua; JZ, Junction Zone; Lab, labyrinth; H&E, hematoxylin and eosin staining; RBC, red blood cells; STB, syncytiotrophoblast. Data present as X ± SEM; NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus WT group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 versus NC-LV group.

Article Snippet: The membranes were blocked with 5% skim milk for 2 h at room temperature, and then incubated overnight at 4°C with primary antibodies against GRK2 (1:1,000, mouse; Santa Cruz, CA, United States), CC3 (1:1,000, mouse; Proteintech Corp., China), Bax (1:1,000, rabbit; BIOSS, China), Bcl2 (1:1,000, mouse; Santa Cruz, CA, United States), RIPK1 (1:1,000, rabbit; BIOSS, China), RIPK3 (1:1,000, rabbit; BIOSS, China), phosphorylated MLKL (p-MLKL) at serine 358 (1:1,000, mouse; BIOSS, China), and β-actin (1:5,000, mice; Proteintech, China).

Techniques: Staining, Knockdown, Quantitative Proteomics, RNA Sequencing, Western Blot, Control, TUNEL Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Role of GRK2 in Trophoblast Necroptosis and Spiral Artery Remodeling: Implications for Preeclampsia Pathogenesis

doi: 10.3389/fcell.2021.694261

Figure Lengend Snippet: Knockdown or inhibition of GRK2 induces RIPK1/RIPK3-dependent necroptosis in HTR8/SVneo cells. (A) HTR8/SVneo cells are transfected with non-targeted siRNA (NC-siRNA) or siRNA targeted to GRK2 (GRK2-siRNA) for 48 h. Representative western blot analysis of cleaved-caspase 3, RIPK1, RIPK 3, phospho-MLKL (p-MLKL; S358), and β-actin (loading control). (B) Cell viability is assessed using CCK8 assay after transfection with siRNA for 48 h. Values are expressed as Mean ± SEM. (C) Pharmacologic inhibition of GRK2 in HTR8/SVneo cells by GSK180736A at different concentrations (0.5, 1, 2.5, and 5 μM) for 12 h. Representative western blot analysis of cleaved-caspase 3, RIPK1, RIPK 3, phospho-MLKL (p-MLKL; S358), and β-actin (loading control). (D) Cell viability is assessed using CCK8 assay after administration with GSK180736A for 12 h. Values are expressed as Mean ± SEM. (E) Flow cytometry with Annexin V/PI-combined staining shows early apoptosis (Ann V + /PI−), and late apoptosis/necrosis (Ann V + /PI +). The rate of late apoptotic/necrotic cells (Ann V + /PI +) is increased significantly after knockdown by GRK2-siRNA, *** P < 0.001. (F) To re-validate the necrosis rate induced by siRNA treatment, loss of mitochondrial membrane potential (ΔΨm) is measured by flow cytometry using JC-1 mitochondrial probes. (G) HTR8/SVneo cells transfected with GRK2-siRNA were examined for nuclear envelope integrity using Lamin B1 antibodies (red; nuclear envelope), or Hoechst (blue; nuclear context), at the indicated time points. Arrows indicate envelope breaches (Lamin B1 staining) and nuclear DNA leakage (Hoechst). Scale bar, 10 μM. (H) TEM images of EVTs treated with/without GSK180736A (cells from villi explants). EVTs on matrigel were necrotic after administration with GSK180736A. From upper to bottom: 8,000×, 20,000× magnification. (I,J) HTR8/SVneo cells treated with/without GSK180736A were collected and subjected to coimmunoprecipitation (Co-IP) with anti-RIPK1 antibody and anti-RIPK3 antibody. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus blank group; ## P < 0.01, #### P < 0.0001 versus NC-siRNA group.

Article Snippet: The membranes were blocked with 5% skim milk for 2 h at room temperature, and then incubated overnight at 4°C with primary antibodies against GRK2 (1:1,000, mouse; Santa Cruz, CA, United States), CC3 (1:1,000, mouse; Proteintech Corp., China), Bax (1:1,000, rabbit; BIOSS, China), Bcl2 (1:1,000, mouse; Santa Cruz, CA, United States), RIPK1 (1:1,000, rabbit; BIOSS, China), RIPK3 (1:1,000, rabbit; BIOSS, China), phosphorylated MLKL (p-MLKL) at serine 358 (1:1,000, mouse; BIOSS, China), and β-actin (1:5,000, mice; Proteintech, China).

Techniques: Knockdown, Inhibition, Transfection, Western Blot, Control, CCK-8 Assay, Flow Cytometry, Staining, Membrane, Co-Immunoprecipitation Assay

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Abnormal retinal development in cloche mutant zebrafish

doi: 10.1002/dvdy.24322

Figure Lengend Snippet: Increased retinal cell death during late retinal neurogenesis in clo mutant embryos. A-D. Immunofluorescence images of wild-type (A,C) and clo−/− (B, D) retinas stained with anti-cleaved caspase 3 (CC3); samples obtained at 54 hpf (A,B) and 72 hpf (C,D). Arrow in B indicates examples of CC3+ cells. E. Numbers of CC3+ and TUNEL+ cells are significantly (***, p<0.001; **, p<0.01) increased in clo−/− retinas at 54 hpf and 72 hpf. F. Numbers of CC3+ (dying) cells are significantly (***; p<0.001) increased in clo−/− retinas at 54 hpf and 72 hpf, but not in clo−/− brains. Bar in A (applies to all images) = 50 μm; LE, lens; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer.

Article Snippet: The following antibodies were used: mouse monoclonal ZPR1, labels red- and green-sensitive cones ( Larison and Bremiller, 1990 ) (1:500; Zebrafish International Research Center; ZIRC); mouse monoclonal ZN8, labels embryonic retinal ganglion cells ( Hu and Easter, 1999 ) (1:500; ZIRC); mouse monoclonal ZRF1, labels Müller glia ( Marcus and Easter, 1995 ) (1:20; ZIRC); rabbit polyclonal anti-GFP (1:1000; Torrey Pines Biolabs), mouse monoclonal 1D1, labels rod opsin ( Fadool, 2003 ) (1:100; the gift of James Fadool, Florida State University); mouse monoclonal anti-HuC/D, labels RGCs and amacrine cells (1:200; Developmental Studies Hybridoma Bank); rabbit polyclonal anti-protein kinase C (PKC), labels a subpopulation of retinal bipolar cells (1:200; Santa Cruz Biotechnologies); mouse monoclonal anti-phosphohistone 3 (PH-3; 1:1000; Cell Signaling Technologies); mouse monoclonal anti-synaptic vesicle 2 (SV2; 1:2000; Developmental Studies Hybridoma Bank); mouse monoclonal anti-cleaved caspase 3 (CC3; 1:200; Abcam); mouse monoclonal 4C4, labels microglia ( Raymond et al., 2006 ) (1:200; gift of Peter Hitchcock, University of Michigan).

Techniques: Mutagenesis, Immunofluorescence, Staining, TUNEL Assay