Nucleic Acid Staining. Fluorophores & Applica6ons

Transcription

1 Nucleic Acid Staining Fluorophores & Applica6ons

2 Types of Nucleic Acid Stains Intercala)ng dyes- - ethidium bromide and propidium iodide Minor- groove binders- - DAPI and the Hoechst dyes Miscellaneous- - nucleic acid stains, including acridine orange, 7- AAD, LDS 751 and hydroxys6lbamidine, with special proper6es.

3 Cell- Impermeant Ethidium Bromide and Propidium Iodide Ethidium bromide and PI are structurally similar phenanthridinium intercalators. PI is more soluble in water and less membrane- permeant than EtBr, although both dyes are generally excluded from viable cells.

4 Ethidium Bromide (EtBr) and Propidium Iodide (PI) EtBr and PI are cell- impermeant; they are excluded from living cells. These dyes intercalate in between the base pairs of the DNA molecule. They bind with limle or no sequence preference at a stoichiometry of one dye per 4 5 base pairs of DNA. Both EtBr and PI also bind to RNA, requiring treatment with nucleases to dis6nguish between RNA and DNA PI is commonly used as a nuclear or chromosome counterstain and as a stain for dead cells.

5 DNA- Selec)ve Hoechst Dyes The Hoechst dyes 33258, and 34580, are cell membrane permeant, minor groove binding DNA stains that are excited by UV light and fluoresce bright blue upon binding to DNA. Hoechst dyes exhibit rela6vely large Stokes shi[s (excita6on/ emission maxima ~350/460 nm), making them suitable for mul6color labeling experiments. They are used in many cellular applica6ons, including cell- cycle and apoptosis studies and they are common nuclear counterstains.

6 AT- Selec)ve DAPI DAPI (4',6- diamidino- 2- phenylindole) shows blue fluorescence upon binding DNA and can be also be excited by UV. The blue- fluorescent DAPI stain apparently associates with the minor groove of dsdna, preferen6ally binding to AT clusters. DAPI is an excellent nuclear counterstain, showing a dis6nct banding pamern in chromosomes. Also useful for cell- cycle studies and mycoplasma detec6on.

7 Acridine Orange: A Dual- Fluorescence Nucleic Acid Stain Acridine orange is a dye that interacts with DNA and RNA by intercala6on or electrosta6c amrac6ons. This dye has green fluorescence with an emission maximum at 525 nm when bound to DNA. Upon associa6on with RNA, its emission is shi[ed to ~650 nm (red fluorescence). Useful for RNA/DNA discrimina6on measurements Lysosome labeling Flow cytometry Cell- cycle studies.

8 7- aminoac6nomycin D 7 AAD is a fluorescent intercalator that undergoes a spectral shi[ upon associa6on with DNA. Weakly permeant. GC- selec6ve. Useful for flow cytometry and chromosome banding.

9 Cell Fixa6on Preserving the cellular structures

10 CELL FIXATION The living cell is fluid or in a semi- fluid state, whereas fixa6on produces coagula6on of 6ssue proteins and cons6tuents, a necessary event to prevent their loss or diffusion during cell processing and/or staining. The objec6ve of fixa6on is to preserve cells and 6ssue cons6tuents in as close a life- like state as possible. Fixa6on arrests autolysis, and stabilizes the cellular cons6tuents so that they can withstand the subsequent stages of processing/staining. Fixa6on should also provide for the preserva6on of cell substances and proteins.

11 General Classes of Fixa6ves Aldehydes, such as formaldehyde, glutaraldehyde. Oxidizing agents: metallic ions and complexes, such as osmium tetroxide, chromic acid. Protein- denaturing agents, such as ace6c acid, methyl alcohol (methanol), ethyl alcohol (ethanol).

12 METHYL AND ETHYL ALCOHOL Protein- denaturing agents These fixa6ves can alter the structure of proteins, primarily due to the disrup6on of the hydrophobic bonds which contribute the ter6ary structure of proteins. However, hydrogen bonds appear to be more stable in methanol and ethanol than in water so that while affec6ng the ter6ary structure of proteins, these alcohols may preserve their secondary structure. These alcohols are both closely related in structure to water and can compete almost as effec6vely as the lamer for hydrogen bonds. Both replace water molecules in the 6ssues, unbound as well as bound, during fixa6on. While absolute ethanol preserves glycogen, it can cause distor6on of the nucleus and shrinkage of cytoplasm. If fixa6on is prolonged, the alcohols remove histones from the nuclei and later extract RNA and DNA.

13 ALDEHYDES Cross- linking fixa6ves The aldehydes form cross- links between proteins, crea6ng a gel, thus retaining cellular components in a life- like state. Soluble proteins are fixed to structural proteins and rendered insoluble, giving some mechanical strength to the en6re structure which enables it to withstand subsequent processing. With the aldehydes, cross- links are formed between protein molecules, the reac6on being mostly with basic amino acid lysine. A disadvantage may be the extent of protein denatura6on produced by this type of fixa6on. This can be cri6cal in the detec6on of an6gens both by immunofluorescence and immunoenzyme techniques.

14 OXIDIZING AGENTS Metallic ions and complexes The most commonly used metallic ion in fixa6on is osmium tetroxide which was ini6ally a 6ssue fixa6ve used in cytology, but poor penetra6on limited its applica6on in light microscopy. Recent studies show that the reac6on of osmium tetroxide is largely with lipid rather than protein. Osmium tetroxide is used for preserva6on of fine structures in electron microscopy and is effec6ve for small (2-3 mm 3 ) specimens.