Sigmafast dab with metal enhancer

Confluent HESC cultures in 4-well chamber slides were decidualized as described above in the presence or absence of 100 µM resveratrol for 4 or 8 days. Following treatment, cells were fixed with 4% buffered paraformaldehyde, endogenous peroxidase activity blocked with 1% H₂O₂ and probed with anti-SIRT1 antibody at a 1:100 dilution overnight (Abcam, Cambridge, UK). Excess primary antibody was washed with TBS-T and probed with a secondary anti-rabbit antibody at a 1:300 dilution (DAKO, Glostrup, Denmark). The expression of SIRT1 was detected using horseradish peroxidase-conjugated streptavidin, 1:300 (DAKO) and visualized with SIGMAFAST™ DAB with Metal Enhancer (Sigma-Aldrich), in which Cobalt chloride was added to enhance 3,3′-diaminobenzidine reaction. Eosin was used as a cytoplasmic counterstain. Images were taken using Keyence BZ-X700 microscope with 40×, 100×, or 200× magnifications (Keyence, Osaka, Japan).

Seeds kept at −80°C were hand dissected whilst frozen and immediately fixed in 0.1 M phosphate buffer pH 7.2 plus 4% paraformaldehyde, 0.1% glutaraldehyde. Samples were then dehydrated in an ethanol series and embedded in Paraplast (Sigma). Sections 8 μm thick were attached to sylanized glass slides. Slides were deparaffinised with xylene (Dimethyl-benzene) and then rehydrated in an ethanol series. Endogenous peroxidase activity was deactivated by incubation in 0.3% hydrogen peroxide for 20 min. Sections were then blocked with 2% normal donkey serum for 2 h at room temperature (RT) and reacted with antiBETL1 or antiBETL2 antisera, or the corresponding pre-sera at 1:500 dilution. Reacted primary antibodies were detected with biotin-conjugated anti-rabbit goat antibody diluted 1:750 (Sigma) and then with Extravidin-peroxidase (Sigma) solution at 1:800 dilution. Finally, positive reaction was developed using SIGMAFAST™ DAB with Metal Enhancer (Sigma) until a gray-black precipitate was clearly visible on the sera-reacted slides. The sections were stained post-detection with 0.025 % Azure B in phosphate buffer pH4 for 3 min, washed in abundant distilled water, mounted in DEPEX and photographed as described in Muñiz et al. (2006 (link)).

The protein in 3 μl of mouse serum was precipitated with ice-cold 75% acetone, and the pellet was solubilized in 100 μl of Laemmli sample buffer. Five microliters of solubilized sample (equivalent to 0.15 μl of serum) was separated on a 4 to 15% TGX gradient gel (Bio-Rad), transferred to a polyvinylidene difluoride (PVDF) membrane, and then probed with biotinylated lectins (Sambucus nigra [SNA], Phaseolus vulgaris erythroagglutinin [E-PHA], Phaseolus vulgaris agglutinin [L-PHA], and Aleuria aurantia [AAL] lectins [Vector Laboratories, Burlingame, CA]) after blocking with 3% BSA in TBS. Probed membranes were incubated with Vectastain ABC-AP (Vector Laboratories), and bound lectin was detected with BCIP/NBT (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Alternatively, PVDF membranes were incubated with anti-inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) antibody (ab180139; Abcam, Cambridge, MA), anti-mouse IgM conjugated to horseradish peroxidase (HRP) (A8786; Sigma), anti-ITIH4 antibody (sc-515353; Santa Cruz), goat anti-rabbit IgG conjugated to AP (Promega, Madison, WI), and rabbit anti-mouse IgG conjugated to AP (Promega, Madison, WI). Incubation with secondary antibodies was followed by detection with SigmaFast DAB with metal enhancer (Sigma, Saint Louis, MO) or incubation with the BCIP/NBT phosphatase substrate as appropriate.

The samples have been prepared in the same way as described above for the detection of the TrwB/VirD4 expression. The only difference being that the expressed proteins were visualized by incubating the membrane with anti‐FLAG antibody produced in rabbit (Abcam) followed by incubation with anti‐rabbit antibody conjugated with horseradish peroxidase (Abcam). The bands were visualized by incubating the membrane with SIGMAFAST™ DAB with metal enhancer (Sigma‐Aldrich).

Seeds kept at −80°C were hand dissected whilst frozen and immediately fixed in 0.1 M phosphate buffer pH 7.2 plus 4% paraformaldehyde, 0.1% glutaraldehyde. Samples were then dehydrated in an ethanol series and embedded in Paraplast (Sigma). Sections 8 μm thick were attached to sylanized glass slides. Slides were deparaffinised with xylene (Dimethyl-benzene) and then rehydrated in an ethanol series. Endogenous peroxidase activity was deactivated by incubation in 0.3% hydrogen peroxide for 20 min. Sections were then blocked with 2% normal donkey serum for 2 h at room temperature (RT) and reacted with antiBETL1 or antiBETL2 antisera, or the corresponding pre-sera at 1:500 dilution. Reacted primary antibodies were detected with biotin-conjugated anti-rabbit goat antibody diluted 1:750 (Sigma) and then with Extravidin-peroxidase (Sigma) solution at 1:800 dilution. Finally, positive reaction was developed using SIGMAFAST™ DAB with Metal Enhancer (Sigma) until a gray-black precipitate was clearly visible on the sera-reacted slides. The sections were stained post-detection with 0.025 % Azure B in phosphate buffer pH4 for 3 min, washed in abundant distilled water, mounted in DEPEX and photographed as described in Muñiz et al. (2006 (link)).