Immunolabeling & Fluorescent Microscopy Presented by: Sumble Maha Khan ABE Workshop June 13 – 30, 2006

Fluorescence is the bombardment of a pigment with high energy light (i.e. blue or UV), which in turn excites the pigment and emits light at a lower E and longer wavelength. What is fluorescence?

Goals of... Immunolabeling : To mark biological molecules or structures using antibody-antigen complexes. For example, the localization of proteins in cells. Fluorescent Microscopy : Is used to obtain a signal from the fluorescent probe on the antibody. For example, AlexaFluor 488.

Immunolabeling ( using the two-step indirect method ) Sections of Arabidopsis provided on slides. Pretreatment with glycine to keep autofluorescence to a minimum. Series of washes & blockings performed to reduce the non-specific binding sites. Overnight incubation with 1 o antibody (anti-CNGC or anti-PDI-2). Washes with TBST + 5% milk. 1 hr incubation in dark with 2 o antibody which has AlexaFluor 488 covalently attached to it. Wash with TBS and place a wet coverslip with plain Vectashield mounting medium. Seal and view under microscope.

Epi-fluorescence Microscope Light source is a mercury arc lamp, has a broad band of excitation wavelengths. Distributes single molecular species based on fluorescence emission properties. Monitors precise location of intracellular components labeled with fluorophores. Study factors such as pH, refractive index, ionic concentrations, membrane potential, solvent polarity.

Confocal Microscopy for high resolution images Light source is a high-intensity monochromatic laser, which excites the fluorophore. Light source is a high-intensity monochromatic laser, which excites the fluorophore. Minimizes background information so image does not degrade. Minimizes background information so image does not degrade. Controls depth of field (z-axis). Controls depth of field (z-axis). Spatial filtering eliminates the out-of-focus light or glare in specimens. Spatial filtering eliminates the out-of-focus light or glare in specimens. Collects serial optical sections of thick specimens, and constructs 3-D images using computer software. Collects serial optical sections of thick specimens, and constructs 3-D images using computer software.

PDI-2 & CNGC PDI-2 ~ protein disulfide isomerase It is one of the PDI family members. It facilitates protein folding by forming disulfide bonds. CNGC ~ cyclic nucleotide gated channel It regulates potassium ion transport in the cells, and it is located in the plasma membrane.

PDI-2 40x 1 o ab ~ 1:10 anti-PDI-2 1 o ab ~ 1:100 anti-PDI-2 2 o ab ~ 1:100 AlexaFluor488 2 o ab ~ 1:100 AlexaFluor 488

PDI-2 40x PDI-2 40x 1 o ab ~ 1:10 anti-PDI-2 1 o ab ~ 1:100 anti-PDI-2 2 o ab ~ 1:10 AlexaFluor o ab ~ 1:100 AlexaFluor 488

PDI-2 40x PDI-2 40x glycine pretreatment no glycine pretreatment 1 o ab ~ 1:10 anti-PDI-2 1 o ab ~ 1:10 PDI-2 2 o ab ~ 1:10 AlexaFluor o ab ~ 1:100 AlexaFluor 488

PDI-2 40x PDI-2 40x 1 o ab ~ 1:100 anti-PDI-2 1 o ab ~ none (control) 2 o ab ~ 1:100 AlexaFluor o ab ~ 1:100 AlexaFluor 488

CNGC 40x CNGC 40x 1 o ab ~ 1:10 anti-CNGC 1 o ab ~ 1:100 anti-CNGC 2 o ab ~ 1:100 AlexaFluor 488

CNGC CNGC 1 o ab ~ 1:10 anti-CNGC 2 o ab ~ 1:100 AlexaFluor 488

CNGC CNGC 1 o ab ~ 1:10 anti-CNGC 1 o ab ~ 1:10 anti-CNGC 2 o ab ~ 1:100 AlexaFluor 488

CNGC 40x CNGC 40x 1 o ab ~ 1:10 anti-CNGC 1 o ab ~ 1:100 anti-CNGC 2 o ab ~ 1:10 AlexaFluor o ab ~ 1:100 AlexaFluor 488

CNGC 40x CNGC 40x 1 o ab ~ 1:100 anti-CNGC 1 o ab ~ none (neg. control) 2 o ab ~ 1:100 AlexaFluor o ab ~ 1:10 AlexaFluor 488

Confocal Images Root 10x Confocal Images Root 10x Arabidopsis root ~ WT (neg. control) Arabidopsis root ~ GFP-2SC (pos. control)

Confocal Images Cotyledon 10x Arabidopsis cotyledon ~ WT Arabidopsis cotyledon ~ GFP-2SC (neg. control) (pos. control)