For transmission electron microscopy, platelets from one representative pPD-L1 high expressing NSCLC patient were used. Platelets were centrifuged and the resulting pellets were fixed for 24 h in Karnovsky’s fixative. As previously described, Ultrathin sections were examined with a LIBRA 120 (Zeiss) operating at 120 kV46 (link). For immunoelectron microscopy, platelets were fixed and embedded in Lowicryl K4M (Polysciences)47 (link). Samples were stained with anti-PD-L1 antibody (Abcam) and examined using a LIBRA 120 transmission electron microscope (Zeiss) at 120 kV.
Lowicryl k4m
Manufactured by Polysciences
Sourced in United States, Germany
Lowicryl K4M is a hydrophilic acrylic resin used for embedding and sectioning of biological specimens for electron microscopy. It is a medium-viscosity resin that can be used for both low-temperature and room-temperature embedding techniques.
Automatically generated - may contain errors
Lab products found in correlation
Libra 120, by Zeiss (2 mentions)
Anti pd l1 antibody, by Abcam (1 mentions)
Tecnai g2 transmission electron microscope, by Thermo Fisher Scientific (1 mentions)
Orius sc200 ccd camera, by Ametek (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Af1556, by R&D Systems (1 mentions)
6 protocols using lowicryl k4m
1
Ultrastructural Visualization of PD-L1 in NSCLC Platelets
2
Immunolocalization of PKP2 in Dog Skin
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Dog skin samples fixed in 4% paraformaldehyde with 0.2% glutaraldehyde were embedded in Lowicryl® K4M (Polysciences; Warrington, PA, USA). Immunolabelling was performed as follows: ultra‐thin, 50 nm sections were blocked in 1% BSA (Sigma‐Aldrich) in PBS for 20 minutes, incubated with primary antibody against PKP2, as shown in the Western blot method given below (Sigma‐Aldrich) in 1:10 dilution in TBS (Tris‐buffered saline) pH 7.4 overnight at 20°C. The sections were washed four times in TBS pH 7.4 and incubated with goat anti‐rabbit antibody conjugated with 20 nm gold particles (115‐205‐144, Jackson Immunoresearch; West Grove, PA, USA) diluted 1:100 in TBS pH 8.3 for 2 h. Next, the sections were washed four times in TBS pH 7.4 and rapidly rinsed in distilled water (dH2O). Thereafter, they were incubated in 5% uranyl acetate for 10 minutes, jet‐washed in dH2O followed by lead citrate 1–2 minutes. After this, the sections were jet‐washed in dH2O and air‐dried. In the negative controls, the primary antibody was omitted. Sections were examined at 80 kV in a Tecnai G2 transmission electron microscope (FEI Company; Eindhoven, Netherlands). Figures were acquired using an ORIUS™ SC200 CCD camera (Gatan Inc.; Pleasanton, CA, USA) using the Gatan Digital Micrograph software.
3
Transmission Electron Microscopy of Bacteria
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Transmission electron microscopy for morphologic analysis of bacteria was performed as previously described [37 (link)]. For immunogold-hBD1red staining 1.2 x 109 CFU/ ml of E. coli were incubated with 200 μg/ ml peptide for 2 hours at 37°C. Bacteria were centrifuged and the pellet was fixed with 3.0% paraformaldehyde, 0.01% glutaraldehyde. The next step was an additional centrifugation and the resulting pellet was embedded in 4% agarose at 37°C and then cooled down to room temperature. Small parts of agarose blocks were embedded in Lowicryl K4M (Polysciences, Germany). Ultrathin sections were cut using an ultramicrotome (Ultracut; Reichert, Vienna Austria). For immunogold labeling the ultra-thin sections (30 nm) were mounted on formvar-coated nickel grids and incubated with rabbit-anti-hBD1red, which was generated in reference [12 (link)]. Finally, the samples were incubated with goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Germany), conjugated with 6 nm gold. For analysis we used a Zeiss LIBRA 120 transmission electron microscope (Zeiss, Oberkochen, Germany) operating at 120 kV.
4
Immunoelectron Microscopy of Mouse Eyes
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Mouse eyes were perfusion-fixed with HBSS, followed by 4% PFA and 0.2% glutaraldehyde in ddH2O via the left ventricle. The eyes were then enucleated, postfixed in 4% PFA and 0.2% glutaraldehyde in ddH2O for 4 hours and subsequently kept in PBS 0.1% PFA at 4°C until further analysis. Fixed eyes were then dehydrated in 30%, 50%, 70%, 95% and 100% ethanol and embedded with lowicryl K4M (Polysciences, Inc, Gröpl), polymerized at minus 35°C and UV light. Ultrathin sections (about 80 nm) were collected on Formvar-carbon coated nickel grids and stained with 2% aqueous uranyl acetate and lead citrate. Immunoelectron microscopy was done essentially as described earlier28 (link). Briefly, Ultrathin sections were blocked with 1% ovalbumin in PBS for one hour, followed by incubation with affinity-purified goat antibody to podocalyxin (R&D, AF1556, 1:400) and an anti-goat 10 nm gold conjugate (1:50). Sections were counterstained with lead citrate. Sections stained with secondary antibody only served as negative controls and did not exhibit any staining.
5
Immunolocalization of RRSV Capsid Protein
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As described in the previous sections, RRSV-infected N. lugens nymphs were
collected at 8 d p.i.; and uninfected nymphs were used as a control. Salivary
glands were dissected and fixed in 4% paraformaldehyde (v/v), 0.3%
glutaraldehyde and 4% sucrose in 0.1 M sodium cacodylate buffer (pH
7.4) for 3 h. After dehydration in serially graded methanol (30, 50,
70, 80, 90, 95 and 100%, v/v), the tissues were embedded in Lowicryl K4M
(Polysciences, Inc., Warrington, PA, USA). Polymerization was performed in an
ultraviolet irradiator at −20 °C for
2 d and then at room temperature for 2 d. Ultra-thin
sections were cut as described for the TEM observations. The sections were
blocked with 1% BSA for 5 min and then incubated with the anti-RRSV
capsid P8 protein mouse serum (1:50) at room temperature for 2 h,
followed by incubation with 5-nm gold-conjugated goat-anti-mouse IgG (1:200,
Sigma-Aldrich) for 2 h. The sections were stained in 3% uranyl
acetate (w/v in distilled water) and observed using TEM with a model JEM-1230 at
an accelerating voltage of 80 kV.
collected at 8 d p.i.; and uninfected nymphs were used as a control. Salivary
glands were dissected and fixed in 4% paraformaldehyde (v/v), 0.3%
glutaraldehyde and 4% sucrose in 0.1 M sodium cacodylate buffer (pH
7.4) for 3 h. After dehydration in serially graded methanol (30, 50,
70, 80, 90, 95 and 100%, v/v), the tissues were embedded in Lowicryl K4M
(Polysciences, Inc., Warrington, PA, USA). Polymerization was performed in an
ultraviolet irradiator at −20 °C for
2 d and then at room temperature for 2 d. Ultra-thin
sections were cut as described for the TEM observations. The sections were
blocked with 1% BSA for 5 min and then incubated with the anti-RRSV
capsid P8 protein mouse serum (1:50) at room temperature for 2 h,
followed by incubation with 5-nm gold-conjugated goat-anti-mouse IgG (1:200,
Sigma-Aldrich) for 2 h. The sections were stained in 3% uranyl
acetate (w/v in distilled water) and observed using TEM with a model JEM-1230 at
an accelerating voltage of 80 kV.
6
Ultrastructural Immunogold Labeling of Platelets
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Platelets were fixed in 3% paraformaldehyde and 0.01% glutaraldehyde for 3 hours at 4°C and then centrifuged. The pellet was embedded in 3.5% agarose at 37°C and cooled on ice. Small parts of agarose blocks were dehydrated in graded ethanol by gradually lowering the temperature to −35°C and embedded in Lowicryl K4M (Polysciences, Germany) at −35°C. The blocks were cut with an ultramicrotome (Ultracut; Reichert, Vienna, Austria), and ultrathin sections (30 nm) were mounted on formvar-coated nickel grids. After the addition of blocking solution (PBS with 10% goat (Dako)), the ultrathin sections were incubated with primary antibodies overnight and secondary antibody for 1 hour (mouse anti-FITC antibody, 1 : 100, Abcam, ab10257). Washing was done with both 1% BSA in PBS and 0.5% skimmed milk powder diluted in 1% BSA in PBS. Subsequently, the sections were incubated with gold-conjugated goat anti-mouse IgG (gold particle diameter: 6 nm; Jackson) and 12 nm gold-conjugated goat anti-rabbit IgG for 1 hour (gold particle diameter: 12 nm; Jackson) (antibodies were diluted at a ratio of 1 : 20 in PBS/BSA/0.5% skimmed milk powder). Finally, the sections were stained with 1% uranyl acetate for 2 minutes. Samples were examined using a Libra 120 electron microscope (Zeiss Oberkochen) operating at 120 k.
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