May gr nwald and giemsa

Samples from the lymph node and bone marrow were collected under general anesthesia, which was carried out with propofol 5 mg/kgbw iv, isoflurane 1.5–2.5 V/V%, and fentanyl by constant rate infusion of 0.01 to 0.04 mg/kgbw/h For routine histological and immunohistochemical examination, an enlarged lymph node was excised, whereas for cytological analysis, bone marrow aspirates were taken with a Jamshidi needle from the iliac crest (crista iliaca externa). For cytological evaluation, the aspirates were smeared and stained with a panoptic staining kit (REAGENS Ltd., Budapest, Hungary).
Sections of (4%, neutralized) formalin-fixed paraffin-embedded lymph node tissues were kept at 56 °C for 12 h in an incubator to prevent the detachment of sections from the silanized slides during antigen retrieval in the immunohistochemistry procedure. The conventional panoptic (May-Grünwald and Giemsa, Sigma-Aldrich, St. Louis, MO, USA) procedure was used to stain the bone marrow.

Bacterial adherence was tested in Hep-2 cells (ATCC CCL-23) monolayers grown to semi-confluence on glass coverslips put in 24 wells microplates (Kasvi, Curitiba, Brazil), filled with 0.7-1.0 ml minimal essential medium Eagle (MEME) (Sigma, St. Louis, USA), supplemented with 5% heat inactivated fetal calf serum (FCS) (Cultilab, Campinas, Brazil), streptomycin, penicillin (Sigma, St. Louis, USA) and amphotericin B (Sigma, St. Louis, USA). Unless otherwise indicated, in all steps below of adhesion and invasion assays, cells incubation was carried out at 37°C in normal (room) concentrations of CO₂ for bacteria, and in 5% CO₂ atmosphere for non-infected or bacteria infected epithelial cells.
After washing the monolayers thrice in phosphate buffered saline (PBS), 0.7-1.0 ml of fresh antibiotic free MEME/FCS containing 1% D-mannose (Sigma, St. Louis, USA) and 20 μl of overnight Luria Bertani (LB) bacterial cultures were added to each well and the plates incubated for 3 hours. Following 3 h incubation, the preparations were washed thrice in PBS, fixed in methanol (Labsinth, Diadema, Brazil) and stained in May-Grünwald and Giemsa (Sigma, St. Louis, USA). The coverslips were mounted on glass-slides and observed under light microscope for attached bacteria and adherence phenotype.

The adherence pattern was determined as described (Santos et al., 2019 (link)) using HeLa cells in 6 h of interaction in the presence of 2% D-mannose (Sigma - USA) to prevent Type 1 fimbria-mediated adhesion. Bacteria previously grown in LB for 18 h at 37°C were inoculated in 1 mL of cell media, at a 1:50 dilution, and incubated at 37°C for 3 h. The preparation was then washed with phosphate-buffered saline (PBS), fresh medium was added, and incubation proceeded for an additional 3 h. After the 6 h period, the cells were washed, fixed with methanol, stained with May Grünwald and Giemsa (Merck, New Jersey, USA) and evaluated by light microscopy (Hernandes et al., 2013 (link)). As controls, the E. coli prototype strains producing the LA (tEPEC E2348/69), AA (EAEC 042), and diffuse adhesion (DA) (DAEC C1845) patterns were used, as well as a non-adherent laboratory strain (E. coli HB101) (Hernandes et al., 2013 (link)).

Cell-culture media, serum, and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA). Bortezomib was provided by Millennium Pharmaceuticals (Cambridge, MA) and melphalan and doxorubicin were obtained from Sigma-Aldrich (St Louis, MO). Annexin-V–FITC was purchased from Immunostep (Salamanca, Spain). May-Grünwald and Giemsa stains were obtained from Merck (Darmstadt, Germany). The origin of the antibodies used in immunocytochemistry and flow cytometry was as follows: anti-CD138-APC (clone B-B4), used for immunocytochemistry and flow cytometry, from Miltenyi Biotec (Auburn, CA); anti-CD20-FITC (clone L27), anti-CD138-PerCP-Cy5 (clone MI15), anti-CD56-APC (clone NCAM16.2), anti-CD45-AmCyan (clone 2D1) and anti-CD38-PE (clone HB7) from BD Biosciences (San Jose, CA, USA); anti-CD19-PacificBlue (clone HIB19) and anti-CD27-PE-Cy7 (clone O323) antibodies from eBioscience (San Diego CA, USA); anti-CD38-AlexaFluor700 antibody (clone HIT2) from Exbio (Vestec, Czech Republic) and anti-CD138-FITC (clone B-A38) from Cytognos S.L. (Salamanca, Spain). The FITC anti-Ki-67 Set was purchased from BD Biosciences (San Diego, CA) and DRAQ5® was obtained from Biostatus (Leicestershire, UK).