Briefly, touch biopsy was performed on a glass slide using a mouse spleen, and then the slide was allowed to dry. Thereafter, the slides were fixed in methanol for 5 min and then stained for 30 min using Giemsa stain (HiMedia, Maharashtra, India). Then, the slides were washed gently and air-dried. The slides were visualized under a light microscope to count the parasites. For all infected and tuzin-immunized groups, the intracellular amastigote parasites were counted in 100 macrophages (35 (link)).
Giemsa stain
Manufactured by HiMedia
Sourced in India
Giemsa stain is a widely used laboratory stain for the identification and differentiation of various blood cells and parasites. It is a mixture of methylene blue, eosin, and azure dyes that stains nucleic acids, proteins, and other cellular components. Giemsa stain is commonly used in the field of hematology, parasitology, and cytology for the purpose of staining and visualizing cellular structures.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Dm il led, by Leica (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Siglec e sirna, by Santa Cruz Biotechnology (1 mentions)
Anti irf3, by Merck Group (1 mentions)
Anti trif, by Novus Biologicals (1 mentions)
Neu1 plasmid dna, by OriGene (1 mentions)
Dynamo colorflash sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions)
Anti tlr4, by Santa Cruz Biotechnology (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Reverse transcriptase kit, by Promega (1 mentions)
Taqman primer, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescein diacetate, by Thermo Fisher Scientific (1 mentions)
Mitotracker red dye, by Thermo Fisher Scientific (1 mentions)
Verapamil, by Merck Group (1 mentions)
Trichloroacetic acid, by HiMedia (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
7 protocols using giemsa stain
1
Giemsa Staining for Parasite Quantification
2
Neuraminidase-Mediated Immune Modulation
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Bovine serum albumin (BSA), anti-phosphotyrosine (cat no-4G10), anti-phospho-IRF3 (cat no-SAB4504031) and anti-IRF3 (cat no-SAB3500280) antibodies were from Sigma (St.Louis, MO). Neuraminidase from Arthrobacter ureafacians (10269611001) was obtained from Roche (Germany). RNeasy Mini Kit was from Qiagen (Limburg, Netherlands); Reverse Transcriptase Kit was from Promega (USA). DyNAmo Color Flash SYBR Green qPCR kit was procured from Thermo Scientific (Rockford, IL). Antibodies-Neu1 (cat no PA5-42552) and Triad3A (cat no-PA5-20079) was from Thermo Fisher Scientific (Rockford, USA). Anti-TLR4 (cat no- MTS510), anti-siglec-E (cat no-377477) antibodies and siglec-E si RNA (cat no-sc-153462) was from Santa Cruz Biotechnology. Anti-Myd88 (cat no-NBP2-27369) and anti-TRIF (cat no-NB120-13810SS) was from Novus Biologicals (Littleton, USA). The cytokine ELISA kits (IFN-β, TNF-α and IL-6) were from Elabscience and from BD-Biosciences (IFN-γ and IL-12). Neu1 plasmid DNA was obtained from Origene (MR226903). All other antibodies were from Cell signaling Technologies (Danvers, MA) unless indicated otherwise. Giemsa stain was obtained from Himedia (India).
3
Characterization of Ayurvedic Formulation YDR
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Yogendra Ras (YDR) (batch number A-YGR011) was procured from Divya Pharmacy, Haridwar, India, sold under its classical name. Based on sensory characterizations, the YDR formulation was observed to be a dry, free-flowing powder and rust-brown in color, packed under inert environment. The YDR powder in dried condition was odorless, tasteless, and insoluble in water and cell culture media due to its metal-based origin under normal physiological pH and temperature. Reagents for cell culture studies such as Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), antibiotics, trypsin-EDTA, isoproterenol, 2′,7′–dichlorofluorescein diacetate (DCFDA), MitoTracker red dye, TaqMan primers and universal RT-PCR master mix for quantitative real-time PCR analysis were purchased from Thermo Fisher Scientific Inc., USA. Purified catalase enzyme was purchased from Merck India Pvt Ltd. Purified superoxide dismutase standards were purchased from Cayman Chemicals, USA. Trichloroacetic acid and Giemsa stain were purchased from Hi-media Laboratories, India. Verapamil was purchased from Sigma-Aldrich, India, and erythromycin was purchased from TCI Chemicals, India.
4
Cytotoxicity Assays of HCT-116 Cells
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DMEM (Dulbecco’s Modified Eagle Medium, Thermo Fisher Scientific, Waltham, MA, USA), Pen Strep (penicillin–streptomycin), PBS (phosphate-buffered saline) and trypsin from Gibco (New York, NY, USA), and FBS (fetal bovine serum) were purchased from Gibco (UK). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). DMSO (dimethyl sulfoxide) and methanol were purchased from Rankem (India). ABTS, gallic acid (≥99%), 5-FU (5-fluorouracil) (≥99%), cisplatin, and ellagic acid (≥95%) were obtained from Sigma (St. Louis, MO, USA). Ethanol, DPPH (1,1-diphenyl-2-picryhydrazyl), crystal violet, and Giemsa stain were purchased from Himedia (Mumbai, India). DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride), Catalog No. D1306 (Eugene, OR, USA); EnzChek™ Caspase-3 Activity Assay Kits, Catalog No. E13183 (Eugene, OR, USA); and CyQuant LDH Cytotoxicity Assay Kit, Catalog No.C20300 (Eugene, OR, USA), were purchased from Invitrogen, Thermo Fisher Scientific (Waltham, MA, USA). HCT-116 cells were obtained from NCCS, Pune, as a kind gift from Dr. Manoj Kumar Bhat.
5
Cisplatin-Induced Blood Cell Morphology Alterations
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The effect of Cisplatin on the blood cell morphology was studied by simple staining of blood smear with gram stain. Blood smear was prepared by placing a single drop of blood on glass slide at a distance of 2 cm from one end and carefully extended to form a uniform smear. The smear was air dried and fixed in 95% ethanol for 10 min. Staining was done using freshly prepared mixture of Giemsa stain (HiMedia lab, Mumbai, India) (0.5 ml of commercial liquid stain diluted in 9.5 ml distilled water) for 30 min. The slides were carefully washed with distilled water, air dried and examined under light microscope equipped with a digital camera (Leica DM IL LED).
6
Arsenic-Induced Blood Cell Morphology in C. striata
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The effect of arsenic on the morphology of blood cells of C. striata was studied by simple staining procedures. Briefly, a single drop of blood was placed on the surface of a clean and grease free microscopic slide at a distance of 2 cm from one end and carefully extended to form a uniform smear. Once prepared, the smear was air dried and fixed in 95% ethanol for ten minutes. Staining was done with a freshly prepared mixture of Giemsa stain (HiMedia, India) (0.5 ml of commercial liquid stain diluted in 9.5 ml distilled water) for 30 min. The slides were carefully washed with distilled water, air dried and examined under light microscope equipped with a digital camera (Leica DM IL LED).
7
Sialyltransferase Silencing Impacts EAEC Adherence
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The effect of silencing of specific sialyltransferase on the adherence of EAEC to each cell line was assessed. Briefly, cells were grown to 50% confluent monolayers, and the media were replaced with antibiotic-free D-mannose (0.5%) containing fresh media. Silencing of each specific sialyltransferase in HCT-15 and INT-407 cell lines was carried out as mentioned earlier. This was followed by EAEC-T8 infection (3 h at 37 °C) of the cells and washing to remove the non-adherent bacteria. The cells were fixed in methanol (70%, 5 min), stained with Giemsa stain (10%, Hi-Media, India), and observed under light microscope for the assessment of EAEC adherence. Also, in parallel, cells under the same conditions were washed with sterile warm PBS to remove non-adherent bacteria followed by lysis in PBS containing 0.5% (v/v) Triton X-100 for 5 min at 37 °C. The suspension containing bacterial cells was serially diluted in PBS and plated on LB agar to quantify the number of colony-forming units (CFU) per ml (Feeney et al. 2017 ). Cells transfected with esi-FLUC-RNA and treated only with lipofectamine 2000 were run as controls.
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