f actin  (Alomone Labs)

Journal: iScience

Article Title: Detection of dedifferentiated stem cells in the Drosophila testis

doi: 10.1016/j.isci.2025.113564

Figure Lengend Snippet: Previous marking method only partially marks dedifferentiated GSCs (A) A schematic diagram illustrating the estimated rates of asymmetric division, symmetric renewal, differentiation, and dedifferentiation of GSCs under steady-state conditions within the testicular niche shown in our previous study. (B) An illustration of the bamGal4-driven FLP lineage tracing system. The bamGal4 driver induces FLP expression in differentiating cells (4-cell and later-stage SGs), where FLP excises the mCherry-stop cassette. If cells with the removed stop cassette revert to the GSC state, they can be identified by the GFP expression under the control of the nosGal4 driver. (C) Representative immunofluorescence images showing the testicular niche in the bamGal4-driven FLP lineage tracing system in the males at 0 and 14 days post-eclosion. The testes were stained with Vasa (blue), FasIII (red), and Hts (red) antibodies. GFP-positive GSCs (green) are encircled by white dotted lines. (D) A graph shows the percentage of GFP-positive GSCs per testis at 0, 7, 14, and 28 days post-eclosion. All scale bars are 10 μm. Asterisks in the images indicate the approximate location of the hub. “n” indicates the number of testes scored.

Article Snippet: The primary antibodies used were as follows: rat anti-Vasa (1:20; developed by A. Spradling and D. Williams, obtained from Developmental Studies Hybridoma Bank (DSHB); mouse anti-hu-li tai shao (Hts) (1:20, 1B1; DSHB) and mouse-anti- Fasciculin III (FasIII) (1:40, 7G10; DSHB).

Techniques: Expressing, IF-cells, Control, Immunofluorescence, Staining

Journal: iScience

Article Title: Detection of dedifferentiated stem cells in the Drosophila testis

doi: 10.1016/j.isci.2025.113564

Figure Lengend Snippet: Reporter#2 cassette is efficiently flipped in GB/SGs but not in GSCs (A) An illustration of the Bam-FLPD5 lineage tracing system. Here, Bam-FLPD5 is combined with the UAS-FRT-stop-FRT-mCD8-GFP cassette and nosGal4 (nos≫mCD8GFP, reporter#2). FLPD5 removes the stop codon from this cassette. If cells once removed stop cassette in the past become GSCs, they can be identified by the GFP expression under the control of the nosGal4 driver. (B) Representative immunofluorescence images of the testicular niche of the Bam-FLPD5 marking system in the males at 0 and 14 days post-eclosion. The testes were stained with Vasa (blue) and FasIII (red) antibodies. GFP-positive GSCs (green) are outlined by white dotted lines. (C) A graph showing the percentage of GFP-positive GSCs per testis at 0, 7, 14, and 28 days post-eclosion. (D) Selected frames from a 12-h time-lapse recording of a niche showing the GFP-activation in two GBs. Germline cells are visualized by the expression of Vasa-mCherry (magenta). White arrowheads indicate GBs becoming GFP positive. The table (left panel) shows the number of times GFP was observed to activate for each indicated cell stage (GSC, GB, and SG) during 12-h live imaging recording of 44 testes. All scale bars are 10 μm. Asterisks in the images indicate the approximate location of the hub. “n” indicates the number of testes scored.

Article Snippet: The primary antibodies used were as follows: rat anti-Vasa (1:20; developed by A. Spradling and D. Williams, obtained from Developmental Studies Hybridoma Bank (DSHB); mouse anti-hu-li tai shao (Hts) (1:20, 1B1; DSHB) and mouse-anti- Fasciculin III (FasIII) (1:40, 7G10; DSHB).

Techniques: IF-cells, Expressing, Control, Immunofluorescence, Staining, Activation Assay, Imaging

Journal: iScience

Article Title: Detection of dedifferentiated stem cells in the Drosophila testis

doi: 10.1016/j.isci.2025.113564

Figure Lengend Snippet: Introduction of an hs-Bam transgene results in a high frequency of marked GSCs (A and B) Representative immunofluorescence images of the testis tip from 7-day-old flies, kept at room temperature, with the Bam-FLPD5 marking system with the nos≫mCD8GFP cassette (reporter#2) alone (A) or with an additional single copy of the hs-Bam transgene (B). Testes were stained with Vasa (blue) and FasIII (red) antibodies. GFP-positive GSCs (green) are outlined with white dotted lines. (C and D) Representative images of live testis tip from 7-day-old flies, kept at room temperature, with Bam-FLPD5 marking system with the nos≫GFP cassette alone (reporter#1) (C) or a combination with a single copy of hs-Bam (D). (E) The graph shows the quantification of GFP-positive GSCs per testis in 7-day-old flies, kept at room temperature, for the indicated genotypes. All scale bars are 10 μm. Asterisks in the images indicate the approximate location of the hub. p -values were calculated by Šídák’s multiple comparisons test. “n” indicates the number of testes scored. (ns: non-significant. p < 0.05: statistically significant).

Article Snippet: The primary antibodies used were as follows: rat anti-Vasa (1:20; developed by A. Spradling and D. Williams, obtained from Developmental Studies Hybridoma Bank (DSHB); mouse anti-hu-li tai shao (Hts) (1:20, 1B1; DSHB) and mouse-anti- Fasciculin III (FasIII) (1:40, 7G10; DSHB).

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Detection of dedifferentiated stem cells in Drosophila testis

doi: 10.1101/2025.03.06.641800

Figure Lengend Snippet: A) A schematic diagram illustrating the estimated rates of asymmetric division, symmetric renewal, differentiation, and dedifferentiation of GSCs under steady-state conditions within the testicular niche shown in our previous study . B) An illustration of the bamGal4-driven FLP lineage tracing system. The bamGal4 driver induces FLP expression in differentiating cells (4-cell and later-stage SGs), where FLP excises the mCherry-stop cassette. If cells with the removed stop cassette revert to the GSC state, they can be identified by the GFP expression under the control of nosGal4 driver. C ) Representative immunofluorescence images showing the testicular niche in bamGal4-driven FLP lineage tracing system in the males at 0 and 14 days post-eclosion. The testes were stained with Vasa (blue), FasIII (red), and Hts (red) antibodies. GFP-positive GSCs (green) are encircled by white dotted lines. D ) A graph showing the percentage of GFP-positive GSCs per testis at 0, 7, 14, and 28 days post-eclosion. E) An illustration of the lineage tracing system where FLP recombinase is directly expressed under a promoter and excises the FRT sites from the reporter cassette. The reporter gene is expressed under a separate promoter. All scale bars are 10 μm. Asterisks in the images indicate the approximate location of the hub. “n” indicates the number of testes scored.

Article Snippet: The primary antibodies used were as follows: rat anti-Vasa (1:20; developed by A. Spradling and D. Williams, obtained from Developmental Studies Hybridoma Bank (DSHB); mouse anti-hu-li tai shao (Hts) (1:20, 1B1; DSHB) and mouse-anti-Fasciculin III (FasIII) (1:40, 7G10; DSHB).

Techniques: Expressing, IF-cells, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Detection of dedifferentiated stem cells in Drosophila testis

doi: 10.1101/2025.03.06.641800

Figure Lengend Snippet: A) An illustration of the Bam-FLPD5 lineage tracing system. Here, Bam-FLPD5 is combined with the UAS-FRT-stop-FRT-mCD8-GFP cassette and nosGal4 (nos>>mCD8GFP). FLPD5 removes the stop codon from this cassette. If cells once removed stop cassette in the past become GSCs, they can be identified by the GFP expression under the control of nosGal4 driver. B) Representative immunofluorescence images of the testicular niche of the Bam-FLPD5 marking system in the males at 0 and 14 days post-eclosion. The testes were stained with Vasa (blue) and FasIII (red) antibodies. GFP-positive GSCs (green) are outlined by white dotted lines. C) A graph showing the percentage of GFP-positive GSCs per testis at 0, 7, 14, and 28 days post-eclosion. D) Selected frames from a 12-hour time-lapse recording (interval:10 min) of a niche showing the GFP-activation in a GB (corresponding to Movie S2). Germline cells are visualized by expression of Vasa-mCherry (magenta). White dotted lines encircle a GB becoming GFP positive. The table (right panel) shows the number of times GFP was observed to activate for each indicated cell stage (GSC, GB, and SG) during 12-hour live imaging recording of 25 testes. All scale bars are 10 μm. Asterisks in the images indicate the approximate location of the hub. “n” indicates the number of testes scored.

Article Snippet: The primary antibodies used were as follows: rat anti-Vasa (1:20; developed by A. Spradling and D. Williams, obtained from Developmental Studies Hybridoma Bank (DSHB); mouse anti-hu-li tai shao (Hts) (1:20, 1B1; DSHB) and mouse-anti-Fasciculin III (FasIII) (1:40, 7G10; DSHB).

Techniques: IF-cells, Expressing, Control, Immunofluorescence, Staining, Activation Assay, Imaging

Journal: bioRxiv

Article Title: Detection of dedifferentiated stem cells in Drosophila testis

doi: 10.1101/2025.03.06.641800

Figure Lengend Snippet: A, B) Representative immunofluorescence images of the testis tip from 7-day-old flies with Bam-FLPD5 marking system with the nos>>mCD8GFP cassette alone (A) or with an additional single copy of the hs-Bam transgene (B) . Testes were stained with Vasa (blue) and FasIII (red) antibodies. GFP-positive GSCs (green) are outlined with white dotted lines. C, D) Representative images of live testis tip from 7-day-old flies with Bam-FLPD5 marking system with the nos>>GFP cassette alone (C) or a combination with a single copy of hs-Bam (D) . E) The graph shows the quantification of GFP-positive GSCs per testis in 7-day-old flies without heat-shock treatment for the indicated genotypes. All scale bars are 10 μm. Asterisks in the images indicate the approximate location of the hub. p-values were calculated by Šídák’s multiple comparisons test. “n” indicates the number of testes scored.

Article Snippet: The primary antibodies used were as follows: rat anti-Vasa (1:20; developed by A. Spradling and D. Williams, obtained from Developmental Studies Hybridoma Bank (DSHB); mouse anti-hu-li tai shao (Hts) (1:20, 1B1; DSHB) and mouse-anti-Fasciculin III (FasIII) (1:40, 7G10; DSHB).

Techniques: Immunofluorescence, Staining