rabbit anti-ph3 primary antibodies  (Millipore)

Journal: BMC Biology

Article Title: The midgut epithelium of mosquitoes adjusts cell proliferation and endoreplication to respond to physiological challenges

doi: 10.1186/s12915-023-01769-x

Figure Lengend Snippet: Cell turnover occurs in the gut epithelium of sugar-fed Anopheles gambiae and Aedes aegypti mosquitoes. Five-day-old females were maintained for 72 h on a diet of 10% sucrose supplemented with EdU prior to dissection. Guts were treated with a Click-iT cocktail to label EdU (green) and stained with an anti-PH3 antibody (red) and DAPI (blue). Cell counts of EdU-positive and PH3-positive cells were obtained for crop, proventriculus (Pv.), midgut (anterior and posterior) and hindgut. Representative images for the whole gut of An. gambiae and Ae. aegypti are shown in ( A ), (scale bar = 500 µm), with a dotted box designating the region of interest used in this study to quantify EdU incorporation (Figs. , , , and ). Magnified images of each region are shown in ( B ), (scale bar = 10 µm). Total counts of PH3-positive ( C ) and EdU-positive cells ( D ) were quantified in each gut region. Results are from at least three biological replicates. Values on top indicate mean values, and error bars are SEM. Three biological replicates were made, and graphs show all replicates combined. Statistics: Mann–Whitney test; *, **, and *** respectively indicate P values of < 0.05, < 0.001, and < 0.001

Article Snippet: All samples were incubated with primary rabbit anti-PH3 antibody (1:500) (Merck Millipore, Darmstadt, Germany).

Techniques: Dissection, Staining, MANN-WHITNEY

Journal: BMC Biology

Article Title: The midgut epithelium of mosquitoes adjusts cell proliferation and endoreplication to respond to physiological challenges

doi: 10.1186/s12915-023-01769-x

Figure Lengend Snippet: The midgut epithelium of adult female mosquitoes shows conserved post-emergence maturation, and dynamic adjustment to changing physiological conditions. Experimental design: A Emerging mosquitoes received EdU in pupal water (maturation experiment). Five-day old mosquitos received EdU in a blood meal (blood feeding experiment), a sucrose-baited suspension of Pseudomonas entomophila (infection experiment) or neither (sugar feeding baseline experiment). All were maintained on 10% sucrose supplemented with EdU for 72 h prior to dissection. Guts were treated with a Click-iT cocktail to label EdU (green) and stained with an anti-PH3 antibody (red) and DAPI (blue). Representative images of the posterior midgut region of interest (ROI) for each condition in all mosquito species are shown in ( B ) (scale bar = 50 µm). To illustrate the amplitude of responses to different stimuli in a single mosquito species, the percentages of EdU-positive cells in the posterior midgut ROI in Aedes aegypti across all conditions are shown in ( C ). Values on top indicate mean values and error bars are SEM. Statistics: one-way ANOVA, P < 0.001. A full graph containing all comparations by species is shown in Fig. S . Percentages of EdU-positive cells in the ROI, relative to the percentages in the sugar-fed condition are shown for maturing ( D ), blood-fed ( E ), and infected ( F ) females of all species. Results are from at least three biological replicates. Values on top indicate mean values, and error bars are SEM. Statistics: Mann–Whitney test; *, **, and *** respectively indicate P values of < 0.05, < 0.001, and < 0.001

Article Snippet: All samples were incubated with primary rabbit anti-PH3 antibody (1:500) (Merck Millipore, Darmstadt, Germany).

Techniques: Suspension, Infection, Dissection, Staining, MANN-WHITNEY

Journal: BMC Biology

Article Title: The midgut epithelium of mosquitoes adjusts cell proliferation and endoreplication to respond to physiological challenges

doi: 10.1186/s12915-023-01769-x

Figure Lengend Snippet: The midgut of a newly emerged female mosquito undergoes a rapid species-specific maturation process characterized by epithelial proliferation and/or endoreplication. The guts of 1, 2, and 3-day old sucrose-fed Aedes aegypti and Anopheles gambiae were dissected immediately following 24 h on a diet supplemented with EdU (administered, respectively, from pupal water to 24 h, 24–48 h, and 48–72 h), and are depicted after Click-iT EdU-labeling (green), anti-PH3 (red) and DAPI (blue) staining. Day 0 guts were dissected within six hours of emergence and stained with DAPI only ( A ) (scale bar = 50 µm). B Quantification of PH3-positive cells revealed higher levels of mitosis at 24 h post-emergence in Ae. aegypti but not An. gambiae . C In both species, EdU incorporation primarily occurred over the first 24 h post emergence. Plots depict the percentage of EdU-positive cells accumulated in the posterior midgut region of interest over 24 h of exposure in 1-, 2-, and 3-day-old mosquitoes, alongside percentages from mosquitoes which received EdU continuously (initially in pupal water, subsequently in sucrose) for 72 h post-emergence (cumulative). Results are three biological replicates. Values on top indicate mean values, and error bars are SEM. Brown-Forsythe and Welch ANOVA (analysis of variance) test for multiple comparisons was utilized. Dunnett’s T3 multiple comparisons test for multiple comparisons; *, **, and *** respectively indicate P values of < 0.05, < 0.001, and < 0.001. D Flow cytometry data from mosquitoes continuously exposed to EdU from the pupal phase showed that the cell populations of dissected posterior midguts at day 3 were significantly altered compared to day 0 in both An. gambiae and Ae. aegypti. Representative histograms are shown. Cell count in the Y-axis is normalized to the total number of cells. Ploidy is indicated at the top of each peak, and gating strategy with diploid controls can be found in Fig. S . E Cell percentage relative to each peak of DNA content was plotted as stacked bar plots, equalizing values to a 100%. F In each cell population, percentages of EdU-positive cells were plotted (median with 95% CI) in stacked bars. Percentages of EdU-positive cells, from each cell population based on nuclei size, show that the cell populations ≥ 32C had the largest percentages of EdU incorporation. For flow cytometry, samples consisted of pools of 40 posterior midguts at day 0 and 25 posterior midguts at day three, n = 9 samples per condition, from at least three biological replicates. Brown-Forsythe and Welch ANOVA (analysis of variance) test for multiple comparisons was utilized to compare EdU positive percentages between ploidy groups. Groups were assigned ID as follows: 2C = a, 4C = b, 8C = c, 16C = d, 32C = e. Letters at the top of each bar indicate which groups are significantly different using Dunnett’s T3 multiple comparisons test for multiple comparisons

Article Snippet: All samples were incubated with primary rabbit anti-PH3 antibody (1:500) (Merck Millipore, Darmstadt, Germany).

Techniques: Labeling, Staining, Flow Cytometry, Cell Counting

Journal: BMC Biology

Article Title: The midgut epithelium of mosquitoes adjusts cell proliferation and endoreplication to respond to physiological challenges

doi: 10.1186/s12915-023-01769-x

Figure Lengend Snippet: Oral infection with Pseudomonas entomophila induces mitosis and ploidy changes in Anopheles gambiae and Aedes aegypti . Mature, 5-day-old females were infected with a sucrose-baited solution containing P. entomophila (OD 600 100) and EdU, thereafter maintained on sucrose/EdU and dissected at 24- or 72-h post-infection (PI). Guts were treated with a Click-iT cocktail to label EdU (green) and stained with an anti-PH3 antibody (red) and DAPI (blue). Representative images of An. gambiae and Ae. aegypti at each timepoint are shown ( A ) (scale bar = 50 µm). For PH3 quantifications ( B ) and 24-h EdU pulse experiments ( C ) midguts were dissected from mosquitoes that were maintained on EdU from 0–24 h (day 1), 24–48 h (day 2) or 48–72 h (day 3) PI. EdU-positive cells were quantified in the posterior midgut region of interest (ROI). Cumulative EdU incorporation in the ROI in unchallenged (UC) mosquitoes and mosquitoes orally infected with P. entomophila /sucrose/EdU and maintained on sucrose/EdU over the full 72 h PI was also quantified. Results are from at least three biological replicates. Values on top indicate mean values. Statistics: Mann–Whitney test; *, **, and *** respectively indicate P values of < 0.05, < 0.001, and < 0.001. Ae. aegypti accumulated the greatest number of EdU-positive cells in the first day, with the rate of incorporation falling on the third day, while the rate of incorporation remained steady in An. gambiae across all three days. This was confirmed also by an EdU/BrdU switch assay, where after the first 24 h PI, mosquitoes were switched from EdU (green) to BrdU (red) ( D ). Most of the cells in the Ae. aegypti posterior midgut incorporated EdU, confirming that there was a period of intensive DNA synthesis during the first 24 h PI. In An. gambiae, the incorporation of EdU and BrdU was comparable, confirming a more modest but sustained response. Flow cytometry data showed that the cell populations from non-infected mosquitoes (sugar-fed) and infected mosquitoes were significantly different from each other . Representative histograms are shown in ( E ). Cell count in the Y-axis is normalized to the total number of cells. Ploidy is indicated at the top of each peak. Relative percentages of the total number of events were graphed as stacked bar plots to present the portion of each population relative to the total ( F ). Samples consisted of pools of 8 posterior midguts, n = 9 samples per condition, from at least three biological replicates

Article Snippet: All samples were incubated with primary rabbit anti-PH3 antibody (1:500) (Merck Millipore, Darmstadt, Germany).

Techniques: Infection, Staining, MANN-WHITNEY, DNA Synthesis, Flow Cytometry, Cell Counting

Journal: BMC Biology

Article Title: The midgut epithelium of mosquitoes adjusts cell proliferation and endoreplication to respond to physiological challenges

doi: 10.1186/s12915-023-01769-x

Figure Lengend Snippet: The mosquito response to the blood meal is source-dependent. Five-day old mosquitoes were blood fed on different sources of blood: human, bovine (Bos taurus), avian (Gallus gallus) and artificial (Skito-Snack). Blood meals were supplemented with EdU, and blood-fed mosquitoes were maintained on sucrose/EdU for 72 h prior to dissection. Guts were treated with a Click-iT cocktail to label EdU (green) and stained with an anti-PH3 antibody (red) and DAPI (blue). Representative images of the posterior midgut region of interest (ROI) at 72 h post-blood feeding are shown in ( A ), scale = 50 µm. Quantification of the percentage of EdU-positive cells in the ROI in Aedes aegypti after feeding with the four different blood sources ( B ) showed that human and bovine blood induced stronger responses in this mosquito when compared with avian blood or the artificial blood meal. When comparing the responses of different species to the same blood-source ( C-F ), Anopheles gambiae showed the strongest response to all but avian blood ( E ). Results are from at least three biological replicates. Values on top indicate mean values, and error bars are SEM. Statistics: Mann–Whitney test; *, **, and *** respectively indicate P values of < 0.05, < 0.001, and < 0.001

Article Snippet: All samples were incubated with primary rabbit anti-PH3 antibody (1:500) (Merck Millipore, Darmstadt, Germany).

Techniques: Dissection, Staining, MANN-WHITNEY

Journal: BMC Biology

Article Title: The midgut epithelium of mosquitoes adjusts cell proliferation and endoreplication to respond to physiological challenges

doi: 10.1186/s12915-023-01769-x

Figure Lengend Snippet: Human blood triggers rapid DNA synthesis in the midgut epithelium of Anopheles gambiae and Aedes aegypti leading to increases in ploidy. Adult mosquitoes were fed on human blood supplemented with EdU and continuously maintained on a diet of sucrose/EdU for up to 72 h prior to dissection. Guts were treated with a Click-iT cocktail to label EdU (green) and stained with an anti-PH3 antibody (red) and DAPI (blue). Representative images of the posterior midgut region of interest (ROI) at 2, 12, 24 and 72 h post-blood meal (PBM) are shown ( A ), scale bar = 50 µm. B - C Quantification of EdU-positive cells using flow cytometry at 2,12, 24 and 72 h post-blood meal also shows early incorporation of EdU after the blood meal in An. gambiae and Ae. aegypti. D - E Quantification of PH3-positive cells in the posterior midgut after 2, 4, 6, 12, 24, 36, 48 and 72 h did not reveal any timepoint at which proliferation was high enough to account for the DNA synthesis observed in An. gambiae . Results are from at least three biological replicates. Values on top indicate mean values, and error bars are SEM. F Flow cytometry analysis shows that upon blood feeding, larger ploidy cells (> 32C) were generated in An. gambiae , effecting a persistent change to epithelial structure. G Ae. aegypti mosquitoes also showed a significant increase of 16C and > 32C cells, but this effect appeared to be transient as the cell population at 72 h post-blood meal reverted to the ploidy profile observed in the sugar-fed epithelium, with the larger cells generated during the peak of the digestive process appearing to be lost

Article Snippet: All samples were incubated with primary rabbit anti-PH3 antibody (1:500) (Merck Millipore, Darmstadt, Germany).

Techniques: DNA Synthesis, Dissection, Staining, Flow Cytometry, Generated

Journal: Journal of Heredity

Article Title: Differences in Cell Proliferation and Craniofacial Phenotype of Closely Related Species in the Pupfish Genus Cyprinodon

doi: 10.1093/jhered/esz074

Figure Lengend Snippet: Immunofluorescence light sheet imaging visualizes number and location of mitotic cells as indicated by assay for pH3 in the heads of hatching-age Cyprinodon. (A) Regions of the head analyzed for proliferating cells. (B) 3D reconstructions in lateral view of the head for the DAPI channel (gray, stains nuclei), the pH3 channel (red, dividing cells), and both channels merged. Note that pH3 positive cells (red dots) are mainly localized to ventral structures as shown in lateral view. (C, D) Single 2D images in (C) frontal view and (D) lateral view show pH3 cells (red dots) localizing to epithelial and mesenchyme tissues surrounding cartilage elements as exemplified by the ceratohyal. Cartilage cells are identifiable in the DAPI channel (gray) by their widely spaced nuclei indicative of large cuboidal cells. Cartilage elements can be identified by shape, and are outlined by the brightly labeled perichondrium, a dense layer of mesenchyme cells that surrounds cartilage elements. Inset shows region outlined by white box and arrows point to pH3 positive cells in perichondrium. Note also the clusters of pH3 positive cells in jaws (arrow head), especially around lateral edge of jaws as seen in panel C. Labels: bh, basihyal cartilage; br, brain; ch, ceratohyal cartilage; ey, eye; ga, gill arches; lj, lower jaw; pa, pharynx; pf, pectoral fin; uj, upper jaw.

Article Snippet: Cells were labeled for pH3 with primary rabbit anti-pH3 polyclonal antibody (MyBioSource, MBS2517807), and secondary antibody Alexa Fluor 568 goat anti-Rabbit (Thermo Fisher, A11011).

Techniques: Immunofluorescence, Imaging, Labeling

Journal: Journal of Heredity

Article Title: Differences in Cell Proliferation and Craniofacial Phenotype of Closely Related Species in the Pupfish Genus Cyprinodon

doi: 10.1093/jhered/esz074

Figure Lengend Snippet: Cell proliferation varies among species of Cyprinodon in different regions of the head at hatching. Shown are number of pH3 positive cells relative to either surface area of sampled tissue (A) or volume of sampled tissue (B) for the 3 regions of the head sampled plus the index of relative jaw proliferation (see text). Plotted are values for each sample and boxplots. Samples sizes are snail-eater N = 30, omnivore N = 28, scale-biter N = 19. Significance for all post hoc pairwise comparisons (Tukey) is shown above boxplots and corresponds to Supplementary Table S3. Note that levels of proliferation significantly vary between species when sampling the entire head (e.g., head region standardized to surface area) or regions of the head posterior to the jaws (post-jaw subset), but proliferation does not vary in the jaws (jaw subset). ns, not significant. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Cells were labeled for pH3 with primary rabbit anti-pH3 polyclonal antibody (MyBioSource, MBS2517807), and secondary antibody Alexa Fluor 568 goat anti-Rabbit (Thermo Fisher, A11011).

Techniques: Sampling

Journal: Scientific Reports

Article Title: A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models

doi: 10.1038/s41598-018-29169-0

Figure Lengend Snippet: PH3 immunostaining, clearing, and segmentation analysis in nocodazole-treated T47D spheroids. ( a ) Maximum intensity projection images of a 15 µm-thick region in the center of 5 DIV 500 cell untreated and 250 nM nocodazole-treated T47D spheroids with nuclear Hoechst counterstaining and PH3 immunostaining. Image brightness is adjusted to visualize staining. ( b) Centroids of segmented nuclei within a 5 DIV 500 cell untreated and 250 nM nocodazole-treated T47D spheroid. Color represents the average intensity of PH3 immunostaining within the segmented nuclei. ( c) Percent of PH3+ cells following treatment with nocodazole. Control with no primary antibody was maintained (no 1°). ( d) Percentage of PH3+ cells was compared between nuclei located in the outer cell layer and the inner cell layers. For ( c and d ) two-way ANOVA with Tukey post-hoc multiple comparisons was performed. Error bars represent SD, **P < 0.0001, ns = not significant. Single points represent individual spheroids. The number of spheroids analyzed was 11, 26, 13, 14, 9, 8, 11, 12, 14, and 11 for no 1°, 0 nM, 16.125 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1 µM, and 2 µM conditions, respectively.

Article Snippet: Primary rabbit anti-phosphohistone H3 (PH3) antibody (06–570, Millipore Sigma) was diluted in PBS with 0.5 w/v% triton X-100 (PBT) at a dilution of 1:100.

Techniques: Immunostaining, Staining, Control