Immunolabeling & Immunolabeling & Fluorescent Fluorescent MicroscopyMicroscopy

Presented by:Presented by:Sumble Maha KhanSumble Maha Khan

ABE WorkshopABE WorkshopJune 13 – 30, 2006June 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 What is fluorescence?fluorescence?

Goals of . . .Goals of . . .Immunolabeling : Immunolabeling :

To mark biological molecules or To mark biological molecules or structures using antibody-antigen structures using antibody-antigen complexes. For example, the localization complexes. For example, the localization of proteins in cells.of proteins in cells.

Fluorescent Microscopy :Fluorescent Microscopy :

Is used to obtain a signal from the Is used to obtain a signal from the fluorescent probe on the antibody. For fluorescent probe on the antibody. For example, AlexaFluor 488. example, AlexaFluor 488.

ImmunolabelingImmunolabeling ((using the two-step indirect methodusing 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 1o antibody (anti-CNGC or anti-PDI-2).

•Washes with TBST + 5% milk.

•1 hr incubation in dark with 2o 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 Epi-fluorescence MicroscopeMicroscope

• 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 MicroscopyConfocal Microscopy for high for high resolution imagesresolution images

Light source is a high-intensity Light source is a high-intensity monochromatic laser, which excites the monochromatic laser, which excites the fluorophore. fluorophore.

Minimizes background information so Minimizes background information so image does not degrade. image does not degrade.

Controls depth of field (z-axis).Controls depth of field (z-axis). Spatial filtering eliminates the out-of-Spatial filtering eliminates the out-of-

focus light or glare in specimens. focus light or glare in specimens. Collects serial optical sections of thick Collects serial optical sections of thick

specimens, and constructs 3-D images specimens, and constructs 3-D images using computer software. using computer software.

PDI-2 & CNGCPDI-2 & CNGC

PDI-2 ~ protein disulfide isomerasePDI-2 ~ protein disulfide isomerase

It is one of the PDI family members. It It is one of the PDI family members. It facilitates protein folding by forming facilitates protein folding by forming disulfide bonds.disulfide bonds.

CNGC ~ cyclic nucleotide gated CNGC ~ cyclic nucleotide gated channelchannel

It regulates potassium ion transport in It regulates potassium ion transport in the cells, and it is located in the plasma the cells, and it is located in the plasma membrane.membrane.

PDI-2PDI-2 40x40x

1o ab ~ 1:10 anti-PDI-2 1o ab ~ 1:100 anti-PDI-2

2o ab ~ 1:100 AlexaFluor488 2o ab ~ 1:100 AlexaFluor 488

PDI-2PDI-2 40x40x

1o ab ~ 1:10 anti-PDI-2 1o ab ~ 1:100 anti-PDI-2

2o ab ~ 1:10 AlexaFluor 488 2o ab ~ 1:100 AlexaFluor 488

PDI-2PDI-2 40x40x

glycine pretreatment no glycine pretreatment 1o ab ~ 1:10 anti-PDI-2 1o ab ~ 1:10 PDI-2

2o ab ~ 1:10 AlexaFluor 488 2o ab ~ 1:100 AlexaFluor 488

PDI-2 PDI-2 40x40x

1o ab ~ 1:100 anti-PDI-2 1o ab ~ none (control)

2o ab ~ 1:100 AlexaFluor 488 2o ab ~ 1:100 AlexaFluor 488

CNGCCNGC 40x40x

1o ab ~ 1:10 anti-CNGC 1o ab ~ 1:100 anti-CNGC

2o ab ~ 1:100 AlexaFluor 488 2o ab ~ 1:100 AlexaFluor 488

CNGCCNGC

1o ab ~ 1:10 anti-CNGC

2o ab ~ 1:100 AlexaFluor 488

CNGCCNGC

1o ab ~ 1:10 anti-CNGC 1o ab ~ 1:10 anti-CNGC

2o ab ~ 1:100 AlexaFluor 488 2o ab ~ 1:100 AlexaFluor 488

CNGCCNGC 40x 40x

1o ab ~ 1:10 anti-CNGC 1o ab ~ 1:100 anti-CNGC

2o ab ~ 1:10 AlexaFluor 488 2o ab ~ 1:100 AlexaFluor 488

CNGC CNGC 40x40x

1o ab ~ 1:100 anti-CNGC 1o ab ~ none (neg. control)

2o ab ~ 1:100 AlexaFluor 488 2o ab ~ 1:10 AlexaFluor 488

Confocal Images RootConfocal Images Root 10x10x

Arabidopsis root ~ WT (neg. control) Arabidopsis root ~ GFP-2SC

(pos. control)

Confocal Images Cotyledon Confocal Images Cotyledon 10x10x

Arabidopsis cotyledon ~ WT Arabidopsis cotyledon ~ GFP-2SC

(neg. control) (pos. control)