Pelco biowave pro

Tissue samples from mouse brain and skin were fixed in 2% PFA, 2.5% glutaraldehyde in 0.15 M Cacodylate buffer pH 7.4 with 2 mM CaCl₂. Samples of Arabidopsis leaves were fixed in 0.5% PFA, 2.5% Glutaraldehyde in 0.1 M phosphate buffer pH 6.8. After several washes in buffer samples were processed in a Pelco Biowave Pro, (Ted Pella, Inc., www.tedpella.com) with use of microwave energy and vacuum. Briefly, samples were fixed in 1% reduced Osmium in cacodylate buffer with CaCl₂ 7× 2 min with alternating microwave power of 100 W/0 W. This step was repeated once. After two washes in UPW with power of 250 W, samples were stained in 1% Uranyl acetate 7× 1 min with alternating microwave power of 150 W/0 W. After two washes in UPW, samples were dehydrated in series of EtOH, each step 40 s at 250 W without vacuum. In the next steps samples were infiltrated in series of different dilutions of Epon resin: EtOH 7× 3 min each at 250 W with vacuum. Finally, samples were embedded in Epon resin 30 min at 200 W, 2× 45 min at 375 W with no vacuum.

To prepare samples, cells were washed in PBS and then fixed in 2.5% glutaraldehyde (GA) in 200 mM HEPES pH 7.4 for 30 min at room temperature then overnight at 4°C. After several washes in 200 mM HEPES buffer, samples were processed in a Pelco Biowave Pro (Ted Pella, USA) with use of microwave energy and vacuum. Briefly, samples were fixed and stained using a reduced osmium, thiocarbohydrazine, osmium (ROTO)/en bloc lead aspartate protocol (Lerner et al., 2017 (link)). Samples destined for stereological analysis were dehydrated using an ethanol series of 50, 75, 90 and 100% then lifted from the tissue culture plastic with propylene oxide, washed four times in dry acetone and transferred to 1.5 ml microcentrifuge tubes. Samples were infiltrated with a dilution series of 50, 75 or 100% (v/v) of Ultra Bed Low Viscosity Epoxy (EMS) resin to acetone mix and centrifuged at 600 g between changes. Finally, samples were cured for a minimum of 48 h at 60°C before trimming and sectioning.
For sectioning and imaging, ultrathin sections (∼50 nm) were cut with an EM UC7 Ultramicrotome (Leica Microsystems) using an oscillating ultrasonic 35° diamond Knife (DiaTOME) at a cutting speed of 0.6 mm/sec, a frequency set by automatic mode and, a voltage of 6.0 volts. Images were acquired using a 120 kV Tecnai G2 Spirit BioTwin (FEI company) with an Orius CCD camera (Gatan Inc.)

CPC and CPC+PCL scaffolds before and after cell seeding and incubation for up to 14 days were imaged via a scanning electron microscope JEOL 7001F (Jeol Ltd., Akishima, Japan) operated at a voltage of 5 kV in a working distance of 8–12 mm, provided by the Science and Engineering Faculty, QUT. For sample preparation, scaffold samples were fixed with 4% formaldehyde (CAS 50-00-0) and glutaraldehyde (CAS 111-30-8) and dehydrated in a PELCO BioWave^® Pro+ (Ted Pella Inc., Redding, CA, USA) using a PELCO Microwave Microcentrifuge Tubes PTFE Holder. In brief, samples were vacuum dried in a series of increasing ethanol concentrations (30–100%; CAS 64-17-5), with additional dimethyl sulfoxide (DMSO; CAS 67-68-5) treatment. Samples were coated with a gold layer using a sputter coater EM SCD005 (Leica Microsystems GmbH, Wetzlar, Germany) at 30 mA for 90 s.

Five honey bees were collected from each of the three colonies maintained in an incubator at 96 h after IAPV infection. The midgut and trachea were dissected and removed as mentioned above, and were prepared for transmission electron microscopy (TEM) as published previously by Pilgrim et al. [32 (link)]. In brief, tissues were dissected into 2% (wt/vol) paraformaldehyde containing 2.5% (wt/vol) glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), which was as fixative. Then, heavy metal staining consisting of ddH₂O was performed using 2% (wt/vol) OsO4, followed by 1% (wt/vol) tannic acid and then 1% (wt/vol) aqueous uranyl acetate. To prevent precipitation, washing the tissue with ddH2O between each staining step was carried out. A Pelco BiowavePro (Ted Pella Inc., Redding, CA, USA) was used to perform fixation and staining steps at 100 W 20 Hg, for 3 min and 1 min, respectively. Dehydration was in a graded ethanol series before filtration and embedding in medium premix resin (TAAB, Reading, UK). For TEM, the tissues were cut into 70 to 74 nm serial sections and collected on Formvar-coated (0.25% (wt/vol) in chloroform; TAAB, Reading, UK) Gilder 200 mesh copper grids (GG017/C, TAAB, Reading, UK) using a UC6 ultra microtome (Leica Microsystems, Wetzlar, Germany). Images were acquired on a 120 kV HT7700 (HITACHI, Tokyo, Japan).

Saliva samples were fixed with glutaraldehyde (2.5%) in a 0.1 M sodium cacodylate buffer for 12 h at 4°C. Saliva samples were deposited in Greiner Bio‐One 96‐well single‐break strip microplates (Greiner Bio‐One) coated with poly‐L lysine to retain the material. Resin embedding of the microplates was then performed as previously described [14 (link)]. Resin embedding was microwave‐assisted with PELCO BiowavePro+ (Ted Pella). Samples were washed with a mixture of 0.2 M saccharose/0.1 M sodium cacodylate and post‐fixed with 1% OsO₄ diluted in 0.2 M potassium hexa‐cyanoferrate (III)/0.1 251 M sodium cacodylate buffer. After being washed with distilled water, samples were gradually dehydrated by successive baths containing 30% to 100% ethanol. Substitution with Epon resin was achieved by incubations with 25%–100% Epon resin, and samples were placed in a polymerization chamber. Resin microwave‐curing was performed for a total of 2 h. After curing, the resin blocks were manually trimmed with a razor blade and the bases of the dishes were detached by cold shock via immersion in liquid nitrogen for 20 s. Resin blocks were placed in a UC7 ultramicrotome (Leica), trimmed to pyramids, and ultrathin 100 nm sections were cut and placed on HR25 300 Mesh Copper/Rhodium grids (TAAB).