Bx53 compound microscope

Polytene chromosome preparation and staining methods were modified from the protocol outlined here^{49 (link)}. A primary antibody against SIN3 (1:1000)^{50 (link)} was followed by a secondary antibody Alexa Fluor 594 (1:400) (Life Technologies). Chromosome spreads were prepared from a minimum of three independent parental crosses and representative images are shown. A minimum of four slides of each control and experimental genotype were prepared at the same time and photographed using identical exposure times. Chromosomes were imaged using an Olympus BX53 compound microscope with a DP72, 12.8 megapixel camera at 400x. Five to ten chromosomal spreads were chosen from each slide for imaging, and images were processed identically using Olympus CellSens software. To quantify SIN3 immunofluorescence signals on polytene chromosomes relative to DAPI intensity, we used a program developed in Matlab 7.4.0 from the protocol outlined here^{51 (link)}. Briefly, batches of control and experimental images, processed identically, are input into the program, which uses two fluorescent channels (DAPI and Alexa Fluor 594). The program applies a mask to each chromosome, removing non-chromosomal antibody staining from the pixel-based quantification.

In detailed anatomical studies, several sections of leaf, and stem were hand-sectioned (~50 µm thick). For uniformity, the middle part of each leaf was sectioned and stained with Toluidine Blue O (TBO) for basic histology observation. Lugol’s iodine was used for starch localization [22 ]. Auramine O was used for lignin and cutin, and fluoral yellow 088 was used for total lipid compounds [23 (link),24 (link)]. All mounts were prepared on glass slides with water. Photomicrographs were obtained using an Olympus BX53 compound microscope equipped an Olympus DP74 camera system (Olympus, Shinjuku, Tokyo, Japan) with fluorescence imaging. Images were processed using OLYMPUS CellSens standard 2 (version 3.1, build 21199) imaging software (Olympus Corp., Tokyo, Japan).

All specimens were kept in 75% ethanol, examined, measured and drawn with an Olympus SZX16 stereomicroscope and an Olympus BX53 compound microscope. Photos were taken with a digital camera Canon PowerShot G12 mounted on an Olympus SZX16 and compound focus images were generated using Helicon Focus software (3.10 Free).
All measurements were given in millimeters. Leg measurements are giving as: total length (femur, patella + tibia, metatarsus, tarsus). The abbreviations used in text including: AER

anterior eye row

; ALE

anterior lateral eyes

; AME

anterior median eyes

; MOA

median ocular area

; PER

posterior eye row

; PLE

posterior lateral eyes

; PME

posterior median eyes

. Specimens are deposited in College of Life Sciences, Hunan Normal University.

For each O₃ exposure time (three, five or seven days), three leaves from the sixth oldest node were used. The medial region of fresh, fully expanded leaf blades was freehand sectioned. Five sections were used for in situ localization of terpenoids using Nadi reagent (naphthol+dimethyl-paraphenylenediamine) [50] . Sections were incubated in the dark for 60 min at room temperature in Nadi reagent, prepared immediately prior to staining. After incubation, the sections were rinsed for 2 min in a sodium phosphate buffer (0.1 M, pH 7.2). By oxidation this reagent forms indophenol blue that changes colour with variation in pH and makes it possible to distinguish between essential oils (blue) and resin acids (intense red), in which a purple color is observed when both compounds constitute the secretion [50] , [51] (link). Five other sections remained untreated – not submitted to any dye or reagents – for observation of the colour and structure of the cells in vivo. Observations and digital images were acquired with an Olympus BX53 compound microscope equipped with an Olympus Q-Color 5 digital camera and Image Pro Express 6.3 software.

hsFLP;Act5C > CD2 > Gal4,UAS-EGFP virgin females were crossed to mCherry^RNAi-TRiP males or UAS-SIN3 complex component RNAi to generate random GFP positive clones via the heat shock flip-out system. Embryos were collected for four hours and then after 48–52 hours, 2^nd instar larvae were subjected to heat shock at 37 °C for two hours. After returning to 27 °C, wandering 3^rd instar larvae were dissected and immunostained 120 hours after egg laying^{19 (link)}. Antibody against GFP (1:1000; Abcam, ab1218) followed by sheep anti-mouse Alexa 488 (1:2000; Life Technologies, A11001) was used for staining. Visualization and imaging was done using an Olympus BX53 compound microscope with a DP72, 12.8 megapixel camera. Images were processed using Olympus CellSens software. Clones were analyzed in a minimum of 20 discs per genotype using Photoshop CS to count the GFP positive pixels for each immunostained disc and comparing that number to the total number of pixels in the DAPI-stained disc^{48 (link)}.

Large sensilla coeloconica (peg organs in pits) on antennal flagella were observed using an Olympus BX53 compound microscope. These sensilla were counted and compared in 36 h post-emerged females. Females from each species were immersed in 10% potassium hydroxide and left for 30–45 min in a hot oven. After clearing, they were washed with 80% ethanol, their antennae were removed using an insect needle and the two antennae of each female were mounted together in Hoyer’s medium on a microscope slide. The large sensilla coeloconica borne on the left and right flagellum of 30 females of each species were counted (n = 60 flagella/species).

Descriptions were made based on specimens fixed in 75% ethanol. The specimens were examined and measured using an Olympus SZX16 stereomicroscope. The details were studied with an Olympus BX53 compound microscope. Male palp and female genitalia were drawn after they were dissected from the spiders. Photos were taken with a Canon PowerShot G12 digital camera mounted on an Olympus SZX16. Compound focus images were generated using Helicon Focus software.
All measurements are given in millimeters. Leg measurements are giving as total length (femur, patella + tibia, metatarsus, tarsus). Abbreviations used are as follows: AER

anterior eye row

; AERW

anterior eye row width

; ALE

anterior lateral eyes

; AME

anterior median eyes

; EL

eye field length

; PER

posterior eye row

, PERW

posterior eye row

; PLE

posterior lateral eyes

. Specimens are deposited in the College of Life Sciences, Hunan Normal University in Changsha, China.