Permount resin

Each skin sample was formalin-fixed and then embedded in paraffin and sectioned at 4 μm. The histological sections were deparaffinized in xylol, rehydrated in ethanol and blocked with 3% hydrogen peroxide. The tissue sections were then incubated with the primary antibodies polyclonal anti-NLRP1 rabbit antibody (Ab 3683); monoclonal mice anti-NLRP3 (214185); polyclonal anti-AIM2 rabbit antibody (93015); polyclonal anti-IL-18 rabbit antibody (Ab71495); and polyclonal anti-IL-1B rabbit antibody (Ab2105), all of which were from Abcam (Cambridge, MA, USA). The Reveal Biotin-Free Polyvalent HRP system (SPB-999, Spring Bioscience Corp, Pleasanton, CA, USA) was selected as the amplification system, and DAB (3,3’ diamibenzidine, D5637, Sigma) was used for the revelation. Negative reaction controls were obtained by omitting the primary antibody and replacing it with PBS pH 7.4. After mounting the slides with Permount resin (FISHER Scientific, Fair Lawn, NJ/USA), the slides were scanned using an Aperio ScanScope slide scanner (Aperio Technologies, Vista, CA, USA). Then, the images were analyzed with Image-Pro Plus, version 4.5.0.29 (Media Cybernetics Inc., Bethesda, MD, USA).

Inflorescences containing flowers at several developmental stages were collected at the Instituto de Biociências of Universidade de São Paulo and at the Instituto de Botânica in São Paulo. Vouchers of the species were provided and the vouchers were deposited in the herbarium of the Universidade de São Paulo (SPF). The material was fixed in formalin, acetic acid, 50% ethyl alcohol (FAA) for 24 h [16 ] or by buffered neutral formalin (BNF) for 48 h [17 ], and then stored in 70% ethyl alcohol.
Inflorescence meristems, flower buds, and anthetic and post-anthetic flowers were isolated, dehydrated in a butyl series [16 ], embedded in Paraplast (Fisher Healthcare, Houston, Texas, USA), and transversely and longitudinally sectioned using a Leica RM2145 rotary microtome (Leica Microsystems, Wetzlar, Germany). Serial sections around 12 μm thick were stained with astra blue and safranin [18 ], and mounted in Permount resin (Fisher Scientific, Pittsburgh, Pennsylvania, USA). Samples were observed and photographed using a Leica DMBL light microscope (Leica Microsystems, Wetzlar, Germany).

Shoot apices of Asclepias, Ipomoea and Urvillea were isolated and fixed in formalin-acetic acid-50% alcohol (FAA) for 24 h (Johansen, 1940 ) and stored in 70% ethanol. Then, the material was dehydrated in an ascending butyl series (Johansen, 1940 ) and embedded in Paraplast (Leica Microsystems, Heidelberg, Germany). All samples were longitudinally sectioned using a Microm HM340E rotary microtome (Microm, Walldorf, Germany) and then stained with 1% astra blue and 1% safranin (Gerlach, 1984 ). Slides were mounted in Permount resin (Fisher Scientific, Pittsburgh, PA, United States) and photographed using a Leica DMLB light microscope.