vectashield antifade mounting medium carrying dapi  (Vector Laboratories)

Journal: Microbiology Spectrum

Article Title: Leishmania donovani elongator protein Elp3a plays a crucial role in modulating the parasite response to genotoxic stress

doi: 10.1128/spectrum.02439-25

Figure Lengend Snippet: elp3a is not essential for survival of Leishmania promastigotes. ( A ) Conserved domains identified in LdElp3a. Boxes demarcate the relevant domains. Numbers indicate the positions of the starting and ending amino acids of the domains. ( B ) Analysis of subcellular localization of LdElp3a-FLAG. Upper panel: Western blot analysis of whole cell lysates isolated from transfectant promastigotes using anti-FLAG antibodies (1:2,500 dil). Tubulin served as loading control. Arrowhead indicates ~85 kDa Elp3a-FLAG protein. Full-length uncropped blots in . Lower panel: microscopic analysis of transfectant promastigotes using immunofluorescence with anti-FLAG antibodies. DAPI stained both the nuclear (N) and kinetoplast (K) compartments. Kinetoplast morphology and segregation pattern was used as cell cycle stage marker. G1/early S: roundish/short rod-like kinetoplast, single nucleus (1N, 1K). Late S/early G2: one elongated kinetoplast, single nucleus (1N,1K). G2/M: one kinetoplast, two nuclei (2N,1K). Post-mitosis: two kinetoplasts, two nuclei (2N, 2K). Magnification bar: 5 µm. ( C ) Analysis of growth of elp3a −/− promastigotes. Cultures were initiated from stationary phase cultures. The experiment was done thrice with technical replicates in each experiment. Values plotted are average of three experiments, and error bars indicate standard deviation. Raw data excel sheets in . ( D ) Analysis of elp3a −/− cell cycle progression. Cells were synchronized at G1/S boundary with 5 mM HU and then released into fresh drug-free medium. Sampling time-points are indicated above the histogram frames. “R” refers to hours after release. Cells in G1, S, and G2M are gated as M1, M2, and M3, respectively. The experiment was done thrice, with comparable results, and one dataset is shown here. The gating strategy is shown in .

Article Snippet: Briefly, Leishmania promastigotes were fixed in 2% paraformaldehyde, cells spread on poly-lysine coated coverslips, permeabilized with 0.1% Triton X-100, blocked with chicken serum (10%), incubated with primary antibody (1:100 FLAG antibody: Cat. no. F1804, Sigma Aldrich, USA), washed and incubated with Texas Red-labeled secondary antibody (1:100, Jackson ImmunoResearch Laboratories, USA), and washed and mounted in Vectashield antifade mounting medium carrying DAPI (VectorLabs, USA).

Techniques: Western Blot, Isolation, Transfection, Control, Immunofluorescence, Staining, Marker, Standard Deviation, Sampling

Journal: Microbiology Spectrum

Article Title: Leishmania donovani elongator protein Elp3a plays a crucial role in modulating the parasite response to genotoxic stress

doi: 10.1128/spectrum.02439-25

Figure Lengend Snippet: Deletion of elp3a has no impact on survival of the parasite within the mammalian host cell. ( A ) Analysis of murine macrophages (J774A.1) infected with Leishmania metacyclics. ( B ) Analysis of human macrophages (THP1 cells) infected with Leishmania metacyclics. Intracellular parasites were scored by mounting the cells in DAPI-containing medium and capturing Z-stack images using a confocal microscope, followed by image analysis using LAS X software. Both experiments were performed thrice, and average values are plotted in the bar graphs. Error bars denote standard deviation. Statistical significance was determined using the student’s t -test. *: P < 0.05, **: P < 0.005, ns: not significant. Raw data Excel sheets in .

Article Snippet: Briefly, Leishmania promastigotes were fixed in 2% paraformaldehyde, cells spread on poly-lysine coated coverslips, permeabilized with 0.1% Triton X-100, blocked with chicken serum (10%), incubated with primary antibody (1:100 FLAG antibody: Cat. no. F1804, Sigma Aldrich, USA), washed and incubated with Texas Red-labeled secondary antibody (1:100, Jackson ImmunoResearch Laboratories, USA), and washed and mounted in Vectashield antifade mounting medium carrying DAPI (VectorLabs, USA).

Techniques: Infection, Microscopy, Software, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Leishmania donovani elongator protein Elp3a plays a crucial role in modulating the parasite response to genotoxic stress

doi: 10.1128/spectrum.02439-25

Figure Lengend Snippet: Analysis of DNA damage in Elp3a-depleted cells subjected to prolonged HU exposure. Microscopic analysis of TUNEL assay reactions carried out on cells that were exposed to 1 mM HU for 24 hours. First row: fluorescein-labeled nuclear and kinetoplast DNA. Second row: DAPI-stained nuclear and kinetoplast DNA. Third row: merged image of fluorescein and DAPI. Log column: untreated cells. 24 h HU column: cells incubated in 1 mM HU for 24 hours. 6.5 hR column: cells incubated in 1 mM HU for 24 hours and then in drug-free medium for a further 6.5 hours. Images were captured by Z stack analysis. Magnification bar: 5 µm. The experiment was performed thrice with comparable results.

Article Snippet: Briefly, Leishmania promastigotes were fixed in 2% paraformaldehyde, cells spread on poly-lysine coated coverslips, permeabilized with 0.1% Triton X-100, blocked with chicken serum (10%), incubated with primary antibody (1:100 FLAG antibody: Cat. no. F1804, Sigma Aldrich, USA), washed and incubated with Texas Red-labeled secondary antibody (1:100, Jackson ImmunoResearch Laboratories, USA), and washed and mounted in Vectashield antifade mounting medium carrying DAPI (VectorLabs, USA).

Techniques: TUNEL Assay, Labeling, Staining, Incubation

Journal: BMC Microbiology

Article Title: Identification of Cdc45-interacting partners uncovers two Leishmania donovani proteins involved in DNA replication

doi: 10.1186/s12866-025-04377-7

Figure Lengend Snippet: Depletion of LdTIM-like prolongs the DNA replication span period of the promastigote population. Cells were pulsed with EdU at various time intervals after release from HU block, and EdU uptake analyzed by Click-iT chemistry coupled to microscopy. (A) Outline of procedure used to assess active DNA replication at various time intervals. (B) Microscopic analysis of EdU uptake in tim-like −/+ and wild type ( tim-like +/+ ) parasites. Panels depict merge of DAPI (blue staining of DNA compartments) and Alexa Fluor 488 (green staining of EdU-labelled DNA) images. White arrowheads indicate EdU-labelled nuclei. Magnification bar: 5 microns (C) Bar chart indicating percent of actively replicating mutant and wild type cells based on percent of cells that are EdU labelled. For each cell type/time-point, 100–125 cells were analyzed. The experiment was performed twice and average values are presented in the bar graph. Error bars indicate standard deviation and statistical significance was determined using the unpaired two-tailed student’s t -test. **: p < 0.005, *: p < 0.05, ns: not significant. HU: EdU pulse after 8 h incubation with 5 mM HU, 1.5 h R: EdU pulse 1.5 h after release from HU-induced block, and so on

Article Snippet: To analyze infections at the two time points (5 h after start of infection and 48 h after start of infection), the macrophages were fixed in 4% paraformaldehyde, permeabilized with 0.25% TritonX-100, and the coverslips were mounted in antifade carrying DAPI (Vectashield, VectorLabs).

Techniques: Blocking Assay, Microscopy, Staining, Mutagenesis, Standard Deviation, Two Tailed Test, Incubation

Journal: BMC Microbiology

Article Title: Identification of Cdc45-interacting partners uncovers two Leishmania donovani proteins involved in DNA replication

doi: 10.1186/s12866-025-04377-7

Figure Lengend Snippet: Depletion of LdPIF6 extends the time taken by the promastigote population to complete DNA replication. Cells were pulsed with EdU at various time intervals after release from HU block, and EdU uptake analyzed by Click-iT chemistry coupled to microscopy (A) Microscopic analysis of EdU uptake in pif6 −/+ and wild type ( pif +/+ ) parasites. Panels depict merge of DAPI (blue staining of DNA compartments) and Alexa Fluor 488 (green staining of EdU-labelled DNA) images. White arrowheads indicate EdU-labelled nuclei. Magnification bar: 5 microns (B) Bar chart indicating percent of actively replicating mutant and wild type cells based on percent of cells that are EdU labelled. For each cell type/time-point, 100–125 cells were analyzed. The experiment was performed twice and average values are presented in the bar graph. Error bars indicate standard deviation and statistical significance was determined using the unpaired two-tailed student’s t -test. *: p < 0.05, ns: not significant. HU: EdU pulse after 8 h incubation with 5 mM HU, 1.5 h R: EdU pulse 1.5 h after release from HU-induced block, and so on

Article Snippet: To analyze infections at the two time points (5 h after start of infection and 48 h after start of infection), the macrophages were fixed in 4% paraformaldehyde, permeabilized with 0.25% TritonX-100, and the coverslips were mounted in antifade carrying DAPI (Vectashield, VectorLabs).

Techniques: Blocking Assay, Microscopy, Staining, Mutagenesis, Standard Deviation, Two Tailed Test, Incubation

Journal: BMC Microbiology

Article Title: Identification of Cdc45-interacting partners uncovers two Leishmania donovani proteins involved in DNA replication

doi: 10.1186/s12866-025-04377-7

Figure Lengend Snippet: Depletion of TIM-like or PIF6 reduces parasite survival in the mammalian host cell. Host macrophages were infected with Leishmania metacylics (wild type or mutant), and infection analyzed at various times thereafter using confocal microscopy. Intracellular parasites were counted by capturing Z-stack images of infected macrophages, followed by counting of DAPI-stained parasite nuclei. The experiment was performed two times and average values are presented in the bar graph. Error bars represent standard deviation. Statistical significance was determined by unpaired two-tailed student’s t -test. *: p < 0.05, ns: not significant

Article Snippet: To analyze infections at the two time points (5 h after start of infection and 48 h after start of infection), the macrophages were fixed in 4% paraformaldehyde, permeabilized with 0.25% TritonX-100, and the coverslips were mounted in antifade carrying DAPI (Vectashield, VectorLabs).

Techniques: Infection, Mutagenesis, Confocal Microscopy, Staining, Standard Deviation, Two Tailed Test