Giemsa staining

Manufactured by Merck Group

Sourced in Germany, United States

Giemsa staining is a laboratory technique used to stain biological samples, particularly blood smears and other cytological preparations. It is a widely used method for the identification and differentiation of different cell types, such as red blood cells, white blood cells, and parasites. Giemsa stain is a complex mixture of methylene blue and eosin dyes, which selectively bind to various cellular components, allowing for the visualization of cellular structures and details.

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Lab products found in correlation

Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions) Neurocult ns a proliferation medium, by STEMCELL (2 mentions) Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions) N2 supplement, by Thermo Fisher Scientific (2 mentions) Epidermal growth factor (egf), by Merck Group (2 mentions) Facsaria fusion instrument, by BD (2 mentions) Axioimager microscope, by Zeiss (2 mentions) Percoll, by Cytiva (2 mentions) Fluo 4 am, by Thermo Fisher Scientific (2 mentions) Masson s trichrome staining, by Merck Group (1 mentions) May gr nwald stain, by Merck Group (1 mentions) 15 ml tube, by Greiner (1 mentions) Trypsin edta, by Merck Group (1 mentions) Methyl methacrylate, by Merck Group (1 mentions) Albumax 2, by Thermo Fisher Scientific (1 mentions) Dmrb light microscope, by Leica (1 mentions) Dfc480 digital camera, by Leica (1 mentions) Dm5500b microscope, by Leica (1 mentions) Rpmi culture medium, by Thermo Fisher Scientific (1 mentions) Hek293e cells, by American Type Culture Collection (1 mentions)

32 protocols using giemsa staining

Bone Defect Histological Analysis

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The harvested bone defect sites were fixed in 10% NBF, decalcified in 0.5 M ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich), dehydrated, and embedded in paraffin. The decalcified bone defect regions were processed for Masson's trichrome staining (Sigma-Aldrich) and hematoxylin and eosin (H &E) staining whereas the undecalcified bone defect regions were processed for Giemsa staining (Merck).

Kubi J.A., Brah A.S., Cheung K.M., Lee Y.L., Lee K.F., Sze S.C., Qiao W, & Yeung K.W. (2023). A new osteogenic protein isolated from Dioscorea opposita Thunb accelerates bone defect healing through the mTOR signaling axis. Bioactive Materials, 27, 429-446.

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Mammary Luminal Progenitor Colony Assay

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For colony-forming assays, the medium used was (human) NeuroCult NS-A Proliferation Medium (StemCell) supplemented with 5% FBS, 10 ηg/ml epidermal growth factor (Sigma), 10 ng/ml basic fibroblast growth factor (Peprotech) and N2 Supplement (Invitrogen) and the cultures were maintained at 37°C/5% CO₂ for 7 days and then fixed using ice cold acetone:methanol (1:1) and visualized using Giemsa staining (Merck). Lin⁻CD24^hiCD49b⁺ luminal progenitors from the flox/flox mammary gland were sorted and plated with irradiated feeders in colony-forming assay medium for 6 days before the number of Ma-CFCs was enumerated

Khaled W.T., Choon Lee S., Stingl J., Chen X., Raza Ali H., Rueda O.M., Hadi F., Wang J., Yu Y., Chin S.F., Stratton M., Futreal A., Jenkins N.A., Aparicio S., Copeland N.G., Watson C.J., Caldas C, & Liu P. (2015). BCL11A is a triple-negative breast cancer gene with critical functions in stem and progenitor cells. Nature Communications, 6, 5987.

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Cytospins and Pappenheim Staining

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To perform cytospins, cells were detached from the culture flasks with a trypsin-EDTA solution (0,05% porcine trypsin; 0,02% EDTA (Sigma-Aldrich) and incubated for 5 min at 37°C. 9 mL FACS buffer was added to block the enzymatic reaction and the detached cell suspension was transferred into a fresh 15 mL tube (Greiner) and centrifuged at 300 g for 5 min 500 μL of the obtained cell suspension containing 100.000 cells were added to a cytofunnel of a cytocentrifuge. Cells were centrifuged at 600 rpm for 5 min to spin the cells down on a slide. The supernatant was discarded and the slides were centrifuged at 1100 rpm for 3 min. Slides were air dried prior to staining. To perform Pappenheim staining, slides were stained for 4 min with May-Grünwald stain (Merck), MilliQ water was added and the slides were incubated for 4 min. Slides were washed with MilliQ water and Giemsa staining (Merck) (1:10) was performed for 10 min. Afterwards the slides were washed with MilliQ water and a coverslip was mounted.

Aktories P., Petry P., Glatz P., Andrieux G., Oschwald A., Botterer H., Gorka O., Erny D., Boerries M., Henneke P., Groß O., Prinz M, & Kierdorf K. (2022). An improved organotypic cell culture system to study tissue-resident macrophages ex vivo. Cell Reports Methods, 2(8), 100260.

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Histological Analysis of Bone Regeneration

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Specimens were fixed in 4% paraformaldehyde and dehydrated in a graded series of ethanol solutions (70, 95, and 100%). Specimens were immersed in xylene and embedded in methyl methacrylate (Merck, Kenilworth, NJ, USA). Specimens were cut into sections (thickness ≈300 mm) by a hard tissue microtome (310 CP Band System; Exakt, Norderstedt, Germany). Five sections of each specimen were produced. Then, a micro grinding system (310 CP; Exakt) was applied to polish the sections down to a thickness of 30–40 mm. Giemsa staining (Merck) was used to observe regenerated osseous tissue under light microscopy (BX53; Olympus).

Li L., Peng X., Qin Y., Wang R., Tang J., Cui X., Wang T., Liu W., Pan H, & Li B. (2017). Acceleration of bone regeneration by activating Wnt/β-catenin signalling pathway via lithium released from lithium chloride/calcium phosphate cement in osteoporosis. Scientific Reports, 7, 45204.

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Mammary Progenitor Cell Colony Assay

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For colony-forming assays, the medium used was (human) NeuroCult NS-A Proliferation Medium (StemCell) supplemented with 5% fetal bovine serum, 10 ηg ml⁻¹ epidermal growth factor (Sigma), 10 ng ml⁻¹ basic fibroblast growth factor (Peprotech) and N2 Supplement (Invitrogen); the cultures were maintained at 37 °C/5% CO₂ for 7 days; then fixed using ice-cold acetone/methanol (1:1) and visualized using Giemsa staining (Merck). Lin⁻CD24^hiCD49b⁺ luminal progenitors from the flox/flox mammary gland were sorted and plated with irradiated feeders in colony-forming assay medium for 6 days before the number of mammary CFCs was enumerated.

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Analyzing Chromosome Separation in Metaphase

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Chromosome preparations from the patient and controls were obtained from lymphocyte cultures of peripheral blood using standard procedures and Giemsa staining (Merck). Metaphase chromosome spreads from the patient and four controls were analyzed. The cells were analyzed under a NIKON E-400 photomicroscope with a 100×/1.30 Plan Fluor oil-immersion objective equipped with a CCD camera model ER-3339 (Applied Imaging Corp.) and the images were processed with the CytoVisionTM version 2.8 software (Applied Imaging Corp.). A total of 100 metaphases were analyzed per cell line and the quantity of cells with chromosome arms open (chromosome arms showed a clear separation) and closed (chromosome arms had no separation) were scored.

Soardi F.C., Machado-Silva A., Linhares N.D., Zheng G., Qu Q., Pena H.B., Martins T.M., Vieira H.G., Pereira N.B., Melo-Minardi R.C., Gomes C.C., Gomez R.S., Gomes D.A., Pires D.E., Ascher D.B., Yu H, & Pena S.D. (2017). Familial STAG2 germline mutation defines a new human cohesinopathy. NPJ Genomic Medicine, 2, 7.

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Airway Inflammatory Cell Analysis in Mice

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Twenty-four hours after the final challenge, BALF was obtained from the mice trachea using an endotracheal tube by instillation and aspiration using 0.4 ml of 1% fetal calf serum (FCS) in PBS for three times (total up to 1.2 ml). The BALF was centrifuged at 350 × g for 5 min at 4 °C, and the supernatant was collected and stored at -80 °C for cytokine analysis, while BALF pellet was used for total cell count. The cell pellets were re-suspended in 100 µl of sterile PBS, and cyto-centrifugation was then carried out using cytospin centrifuge (Thermo Shando, Pittsburgh, USA). The cells were air-dried and stained with methanol for 1–2 min, followed by Giemsa staining (Merck, Germany) for 8 min, and the dried slides were mounted. A minimum of 200 inflammatory cells were identified by standard morphology criteria using a light microscope (Olympus, USA) at 100 × magnification. The relative count number of eosinophil, macrophage, lymphocyte and neutrophil were calculated.

Muhamad S.A., Safuan S., Stanslas J., Wan Ahmad W.A., Bushra S.M, & Nurul A.A. (2023). Lignosus rhinocerotis extract ameliorates airway inflammation and remodelling via attenuation of TGF-β1 and Activin A in a prolonged induced allergic asthma model. Scientific Reports, 13, 18442.

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Antileishmanial Activity of tREP-18 Peptide

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The J774.A1 murine macrophage cell line was plated
at a cell density of 5 × 10⁵ cell well^–1 in a six-well flat bottom plate. For infection, late-stage L. donovani rich in metacyclic promastigotes was
added at a ratio of 10:1 along with the peptide of interest. After
12 h, uninfected promastigotes were washed off with PBS. Infected
macrophages were treated with the tREP-18 peptide at nearly an IC50
concentration and incubated for 72 h. Amphotericin B was taken as
a positive control. After incubation, visualization and counting of
intracellular parasite load were performed using Giemsa staining (Sigma-Aldrich).

Chakrabarti A., Kaushik M., Khan J., Soota D., Ponnusamy K., Saini S., Manvati S., Singhal J., Ranganathan A., Pati S., Dhar P.K, & Singh S. (2022). tREPs—A New Class of Functional tRNA-Encoded Peptides. ACS Omega, 7(22), 18361-18373.

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Immunostaining and FACS Analysis of Fetal Liver Cells

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Fetal liver cells were immunostained with two separate panels (see Supplementary Table 15 for antibody details). Cells were stained for 30 minutes on ice followed by DAPI staining. FACS was performed on a BD FACSAria™ Fusion instrument, and data analysed using FlowJo (v10.4.1). Cells were isolated into chilled FACS tubes coated with FBS and prefilled with 500μL sterile PBS for cytospin (500 – 2000 cells), or into 1.5mL microfuge tubes containing 20μL lysis buffer (100 cells). Giemsa staining (Sigma-Aldrich) was used to determine the morphology of sorted cells on cytospins. Slides were viewed using a Zeiss AxioImager microscope, images taken of 4 fields from n = 3 samples using the 100x objective, and viewed using Zen (v2.3). Raw images supplied in Supplementary Images.

Popescu D.M., Botting R.A., Stephenson E., Green K., Webb S., Jardine L., Calderbank E.F., Polanski K., Goh I., Efremova M., Acres M., Maunder D., Vegh P., Gitton Y., Park J.E., Vento-Tormo R., Miao Z., Dixon D., Rowell R., McDonald D., Fletcher J., Poyner E., Reynolds G., Mather M., Moldovan C., Mamanova L., Greig F., Young M., Meyer K.B., Lisgo S., Bacardit J., Fuller A., Millar B., Innes B., Lindsay S., Stubbington M.J., Kowalczyk M.S., Li B., Ashenbrg O., Tabaka M., Dionne D., Tickle T.L., Slyper M., Rozenblatt-Rosen O., Filby A., Carey P., Villani A.C., Roy A., Regev A., Chedotal A., Roberts I., Göttgens B., Behjati S., Laurenti E., Teichmann S.A, & Haniffa M. (2019). Decoding human fetal liver haematopoiesis. Nature, 574(7778), 365-371.

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Isolation of Murine Neutrophils and Macrophages

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Murine neutrophils and macrophages were isolated from bone marrow of either WT or Ccn1^D125A/D125A mice as described with modifications^{69 , 70 (link)}. For neutrophils isolation, the resuspended bone marrow in Hank’s balanced salt solution (HBSS, 5ml) was carefully layered over 62% Percoll (5 ml, Amersham) and centrifuged at 1000 xg for 30 min. After the gradient centrifugation, a cloudy layer containing neutrophils was removed from on top of the Percoll layer and resuspended in RPMI media for culture. An aliquot of cell suspension (~20 µl) was spread on to slide glass to assess the purity of isolated neutrophils by Giemsa staining (Sigma). For macrophages, the bone marrow cells were re-suspended in macrophage complete media (DMEM/F12 with 20% (v/v) L-929 cells conditioned media), counted, and 4×10⁵ cells were plated in sterile plastic petri dish (100 mm) and incubated in 37°C in 5% CO₂ for 7 days with medium being refreshed on day 3 for full differentiation into macrophages. The macrophages differentiation was checked using F4/80 immunostaining.

Jun J.I., Kim K.H, & Lau L.F. (2015). The Matricellular Protein CCN1 Mediates Neutrophil Efferocytosis in Cutaneous Wound Healing. Nature communications, 6, 7386.

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