Method description for immunolocalization studiesPlant tissue preparation
To examine distribution of IAA, plant materials were embedded in paraplast as described in Lee et al. 2008. The plant materials were cut into small pieces (2 to 3 mm-thick) and immediately fixed in 4% paraformaldehyde in sodium phosphate buffer and 0.1% (v/v) Tween 20 under vacuum for 1 h, and left overnight at 4°C. After fixation, the plant materials were washed in 0.85% (w/v) saline, dehydrated through a graded alcohol series and embedded in paraplast. The plant materials embedded in paraplast sections (10 µm thick) were collected on poly-L-lysine coated slides. Imunolabeling
The plant materials embedded in paraplast sections (10 µm thick) were deparaffinized using histoclear (Cell path, England), rehydrated with an ethanol series, and washed with water. Dewaxed and re-hydrated sections were incubated in 10 mM phosphate-buffered saline (PBS) for 5 min. Blocking
was done using 10 mM PBS, 0.1% (v/v) Tween 20, 1.5% (v/v) Glycine, and 5% (w/v) BSA for 45 min at 20°C. Wash
in 10 mM PBS, 0.88% (w/v) NaCl, 0.1% (v/v) Tween 20, and 0.8% (w/v) BSA) for 5 min and brief wash in10 mM PBS and 0.8% (w/v) BSA. Primary antibody incubation
Agrisera anti-IAA antibody has been used on each slide (100 µl) in dilution 1: 100 and incubated in a humid chamber during overnight at 20ºC. Wash
Two times vigorous wash in 10 mM PBS, 2.9% (w/v) NaCl, 0.1% (v/v) Tween 20, and 0.1% (v/v) BSA for 10 min each were followed by a 10 min wash with 10 mM PBS, 0.88% (w/v) NaCl, 0.1% (v/v) Tween 20, and 0.8% (w/v) BSA and brief rinse with 10 mM PBS and 0.8% (v/v) BSA. Secondary antibody incubation and reaction development
100µl anti-rabbit IgG-alkaline phosphatase-conjugate (Sigma, USA) with 1:100 (w/v) dilution were added to each slide as secondary antibody and incubated overnight in a humidity chamber at 20°C. Two 15 min washes with 10 mM PBS, 0.88% (w/v) NaCl, 0.1% (v/v) Tween 20, and 0.8% (w/v) BSA were followed by a 15 min wash with water. The sections were visualized by applying 200 µl ready-to-use Western Blue (Promega, USA) and incubated in the dark for 15 to 30 min at room temperature (20°C). When blue/purple color was observed, slides were finally washed in water and then dehydrated in ethanol series. Slides were mounted with Depex (BDH, USA). Photographs were taken using Leitz microscope using bright field optics (Leitz, Germany). Negative control was done ommition of primary antibody.
Courtesy Dr Lee
Example of Western blot protocol : Membrane blocking, antibodies incubations and detection
1) Saturate the blot membrane with TBS + 5% Blocker for 1 hour at 37°C while mixing
2) Wash the membrane twice for 5 minutes in TBS Tween at 37°C
3) Incubate the membrane with anti-IAA antibodies diluted 1 : 1000 – 1 : 2000 in TBS 0.5% Blocking solution for 2 hours at 37°C
4) Wash the membrane three times for 5 minutes in TBS Tween at 37°C
5) Incubate with a biotinylated secondary antibody diluted (1: 1000-1: 2000) in TBS 0.5% Blocking solution for 2 hours at 37°C
6) Wash the membrane three times for 5 minutes in TBS Tween at 37°C
7) Incubate with Streptavidin-HRP 1μg/ml in TBS 0.5% Blocking solution for 2 hours at room temperature
8) Wash the membrane three times for 5 minutes in TBS at 37°C
9) Incubate in TBS (200ml) + (50mg DAB in 25ml methanol) + (150mg 4-chloro-1-naphtol in 25ml methanol) + 50μl H2O2 30% for a maximum of 30 minutes in the dark
10) Stop the reaction by addition of distilled water
Blocking solution = skim milk (Biorad 170-6404) TBS = 20mM Tris base, 0.5M NaCl, pH 7.5 TBS Tween = TBS + 0.05% Tween 20
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