Calcofluor white

ClearSee solutions were prepared by mixing xylitol powder [#04; final 10% (w/v)], sodium deoxycholate [#07; final 15% (w/v)] and urea [#19; final 25% (w/v)] in water. Seedlings, leaves and pistils of A. thaliana and gametophores of P. patens were fixed with 4% (w/v) PFA for 30-120 min (seedlings, 30 min; leaves, 120 min; pistil or gametophores, 60 min) in PBS under vacuum (∼690 mmHg) at room temperature. Fixed tissues were washed twice for 1 min each in PBS and cleared with ClearSee at room temperature for 4 days to 4 weeks or more, depending on tissue type. The minimum incubation times for clearing were 4 days for leaves, roots and moss, 7 days for seedlings, 2 weeks for pistils, and 4 weeks for mature stems. In the case of pistils, incubation for 4 weeks improved clarity. ClearSee-treated samples could be stored at room temperature for at least 5 months. For post-staining, cleared tissues were stained with Calcofluor White (final 100 µg/ml) in ClearSee solution for 1 h, and Hoechst 33342 (final 10 µg/ml) in ClearSee solution overnight. After staining, tissues were washed in ClearSee for 1 h.

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.

Tamarind (Tamarindus indica L.) seeds were obtained from Jungle Seeds, Watlington, UK) and nasturtium (Tropaeolum majus L. cv Tom Thumb) seeds from Mr. Fothergill's Seeds Ltd., Newmarket, UK. Tamarind and nasturtium seeds were imbibed for 24 h and then pieces of cotyledon parenchyma were excised, fixed and prepared for embedding in LR White resin with subsequent sectioning for indirect immunofluorescence analysis as described previously [8 (link)]. Tobacco (Nicotiana tabacum L.) and pea (Pisum sativum L.) plants were grown in a greenhouse with 16 h days and maintained between 19 and 23°C. Regions of second internodes from the top of six-week old plants were fixed, embedded in wax and sectioned as described previously [46 (link)].
In addition to LM15, three further monoclonal antibodies were used in this study using indirect immunofluorescence: CCRCM1, a mouse monoclonal antibody to a fucosylated epitope of xyloglucan [19 (link)], a gift from Dr. Michael Hahn (CCRC, University of Georgia, USA), JIM5, a rat monoclonal antibody to methyl-esterified and unesterified epitopes of HG [32 (link)] and LM6, a rat monoclonal antibody to arabinan [34 (link)]. Section pre-treatment to remove HG from cell walls involved incubation of sections with a recombinant microbial pectate lyase 10A [47 (link)] (a gift from Prof. Harry Gilbert, University of Newcastle-upon-Tyne) at 10 μg/mL for 2 h at room temperature in 50 mM N-cyclohexyl-3-aminopropane sulfonic acid (CAPS), 2 mM CaCl₂ buffer at pH 10 as described [10 (link)]. The high pH of the enzyme buffer removes HG methyl esters in cell walls and results in HG being susceptible to pectate lyase degradation and also suitable for recognition by JIM5. Sections not treated with the pectate lyase were incubated for an equivalent time with the high pH buffer without enzyme and imaged as untreated controls. After enzyme or buffer treatment, sections were incubated in phosphate-buffered saline (PBS) containing 5% (w/v) milk protein (MP/PBS) and a 5-fold dilution of antibody hybridoma supernatant for 1.5 h. Samples were then washed in PBS at least 3 times and incubated with a 100-fold dilution of anti-rat IgG (whole molecule), or anti-mouse IgG, linked to fluorescein isothiocyanate (FITC, Sigma, UK) in MP/PBS for 1.5 h in darkness. The samples were washed in PBS at least 3 times and incubated with Calcofluor White (0.2 μg/mL) (Fluorescent Brightner 28, Sigma, UK) for 5 min in darkness. Samples were washed at least 3 times and then mounted in a glycerol-based anti-fade solution (Citifluor AF1, Agar Scientific, UK). Immunofluorescence was observed with a microscope equipped with epifluorescence irradiation and DIC optics (Olympus BX-61). Images were captured with a Hamamatsu ORCA285 camera and Improvision Volocity software.

Conidia were taken in 0.5-ml Eppendorf tubes mixed with 10 μl of calcofluor white stain, vortexed for 10 s, and then incubated for 15 min in the dark. After incubation, conidia were taken in glass slides, sealed on four sides, and visualized in a fluorescence microscope (40 ×) to check the staining efficiency.

The cellulose of the biofilm on the coverslips was washed with PBS three times to remove the planktonic bacteria. The coverslips were then placed at 60 °C for 1 h to fix the biofilm and then stained with 0.1 mg/mL calcofluor white for 20 min in a dark room. After being washed with PBS three times, the biofilm was observed with a fluorescence microscope (SUNNY ICX41, Yuyao, China).