Triethoxysilane

Two flowers per day from anthesis, two and three days after pollination were fixed in 4% formaldehyde freshly prepared from paraformaldehyde in 1x phosphate saline buffer (PBS) pH7.3, left overnight at 4ºC, and conserved then at 0.1% formaldehyde solution [83 (link)]. Then the pistils were dehydrated in an acetone series (30%, 50%, 70%, 90%, 100%), and embedded in Technovit 8100 (Kulzer and Co, Germany) for two days. The resin was polymerized at 4ºC, and sectioned at 4 μm thickness. Sections were placed in a drop of water on a slide covered with 2% (3-Aminopropyl) triethoxysilane - APTEX (Sigma-Aldrich), and dried at room temperature. Callose was identified with the anticallose antibody (AntiCal) that recognises linear β-(1,3)-glucan segments (anti-β-(1,3)-glucan; immunoglobulin G1), Biosupplies, Australia [49 (link)]. As a secondary antibody, Alexa 488 fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG was used (F-1763; Sigma). Additionally, a monoclonal antibody (mAbs) JIM13 [84 (link)] against AGPs glycosyl epitopes, and one mAb JIM11 [85 (link)] against extensin epitopes were obtained from Carbosource Services (University of Georgia, USA). Secondary antibodies were anti-rat IgG conjugated with the same Alexa 488 used above. Sections were incubated for 5 min in PBS pH7.3 followed by 5% bovine serum albumin (BSA) in PBS for 5 min. Then, sections were incubated at room temperature for 1h with AntiCal primary mAb, JIM13, and JIM11. After that, three washes in PBS of 5 minutes each preceded the incubation for 45 min in the dark with a 1/25 diluted secondary fluorescein isothiocyanate (FITC) conjugated with the antibody in 1% BSA in PBS, followed by three washes in PBS [83 (link)]. Sections were counterstained with calcofluor white for cellulose [86 (link)], mounted in PBS or Mowiol, and examined under a LEICA DM2500 epifluorescence microscope connected to a LEICA DFC320 camera. Filters were 355/455 nm for calcofluor white and 470/525 nm for the Alexa 488 fluorescein label of the antibodies (White Level?=?255; Black Level = 0; ϒ?=?1). Exposur (Exp) times were adapted to the best compromise in overlapping photographs for each antibody: AntiCal, Exp.?=?15.30ms (Calcofluor Exp. = 1.20ms); JIM13 Exp.?=?2.52ms (Calcofluor?=?0.41ms); JIM11, Exp. = 31.59 ms (Calcofluor Exp. = 1.40ms). Brightness and contrasts were adjusted to obtain the sharpest images with the Leica Application Suite software.

The tetraethylorthosilicate (TEOS) (98%), (3-aminopropyl)triethoxysilane (APTES) (99%), toluene (99%), and anhydrous ethanol (99.7%) were sourced from Acros, Morris Plains, NJ, USA. The urea (99%), hydrochloric acid (36.5–38.0%), nitric acid (68–70%), aqueous ammonia (25–28%), potassium hydroxide (≥99%), sodium hydroxide (≥99%), and potassium perchlorate (≥99%) were acquired from Fisher Scientific Co., Fair Lawn, NJ, USA. The formaldehyde solution (~37.0%) was obtained from J.T.Baker, Radnor, PA, USA. The uranyl nitrate hexahydrate (UO₂(NO₃)₂∙6H₂O) (≥99%) was ordered through EHS, the University of Delaware, with the SPI from West Chester, PA, USA, as the distributor.

Under a nitrogen shield, 1 g of porous silica was placed into a reactor. Subsequently, 100 mL of toluene (99%) and 1 mL of APTES were sequentially added to the reactor. The solution in the beaker was then agitated and heated at 70 °C for 12 h. The solution was filtered after the reaction, and the resulting residue was collected. The residue was subsequently washed with anhydrous ethanol and then dried at 100 °C for 12 h. This process led to the formation of (3-aminopropyl)triethoxysilane-functionalized porous silica (AP@MPS), which was exhibited as a yellow powder. Figure 15 is at representation of the grafting reaction.