Fig. 1
Effects of histone acetylation and deacetylation through the actions of histone acetyl transferase (HAT) and histone deacetylase (HDAC) respectively. HDAC inhibitors (HDACi) increase acetylation by inhibiting the action of HDAC
Table 1
The 11 metal-dependent HDAC enzyme and their subdivisions
Class | Enzyme | Location | Expression |
|---|---|---|---|
Class I (Rpd3) | HDAC1 | Nucleus | Ubiquitous |
HDAC2 | |||
HDAC3 | |||
HDAC8 | |||
Class IIa (Hda1) | HDAC4 | Nucleus/cytoplasm | Tissue specific |
HDAC5 | |||
HDAC7 | |||
HDAC9 | |||
Class IIb (Hda1) | HDAC6 | Cytoplasm | Tissue specific |
HDAC10 | |||
Class IV (Rpd3/Hda1) | HDAC11 | Nucleus/cytoplasm | Tissue specific |
Systematic organ and disease specific analysis of histone deacetylases therefore offers us a valuable insight into the regulation of kidney disease, and the mechanisms of progression. In this chapter we describe a histochemical based technique to detect and analyze HDAC expression in routinely collected tissue sections.
2 Materials
All aqueous solutions are made in laboratory grade deionized water (dH2O).
2.1 Fixative
1.
0.01 M phosphate buffered saline (PBS): Prepare a 10× stock by dissolving 80 g of NaCl, 2.0 g of KCl, 14.4 g of Na2HPO4, and 2.4 g of KH2PO4 in 900 mL of dH2O. Adjust to pH 7.4 and make up to 1 L. Dilute 1:10 for a 1× working concentration.
2.
4 % PFA: Add 4 g paraformaldehyde (BDH) to 100 mL 0.01 M phosphate buffered saline (PBS) pH 7.4, heat to a maximum of 60 °C with stirring and add 5–10 drops of 1 M NaOH to clear.
2.2 Histological Processing and Tissue Sectioning
1.
Labeled glass vials.
2.
Graded ethanol: 50, 70, 95, and 100 % v/v mixture of ethanol and dH2O.
3.
Chloroform.
4.
Paraplast™ paraffin embedding medium (McCormick Scientific, St. Louis, MO, USA), or equivalent low temperature embedding wax.
5.
Embedding molds and cassettes.
6.
Scalpel and No. 22 blade.
7.
Artist’s paint brush.
8.
Shallow bowl filled with a 20 % v/v mixture of ethanol in dH2O.
9.
Microtome.
10.
Histological water bath.
11.
Slide racks.
13.
Chloroform.
2.3 HDAC Staining
1.
Xylene (or less toxic equivalent such as Histolene™).
2.
Ethanol (100, 90, 70 % v/v in dH2O).
3.
PBST: 0.1 % Tween 20 in PBS (without Ca2+ and Mg2+).
4.
PBST with 1 % bovine serum albumin (BSA) (v/v).
6.
0.1 % Triton™ X-100 (Sigma, St. Louis, MO, USA).
7.
1 % w/v BSA in PBS (without Ca2+ and Mg2+).
8.
Polyclonal rabbit anti-HDAC1-11 antibody panel (Biovision, Milpitas, CA, USA).
9.
Polyclonal goat anti-β-actin (Abcam, Cambridge, UK).
10.
Alexa Fluor™ goat 546 anti-rabbit IgG (H + L) (Invitrogen; Life Technologies, Grand Island, NY, USA).
11.
Alexa Fluor™ donkey 488 anti-goat IgG (H + L) (Invitrogen).
12.
ProLong™ Gold Antifade Solution with DAPI (Invitrogen)
13.
Water-repelling immunostaining pen (e.g., Pap Pen™).
14.
Coverslips.
15.
Nail polish.
16.
Rotating platform.
17.
Slide racks.
18.
Staining jars.
19.
Humidified incubation chamber.
20.
Microwave oven.
2.4 Image Acquisition
1.
Fluorescence microscope, with digital camera (e.g., Olympus BX61 automated upright microscope with FVII camera).
2.5 Heat Map Analysis
1.
Image J analysis software version (1.146d or later) for the appropriate platform (Downloadable as freeware from Fiji Is Just Image J) [12].
2.
Microsoft Excel (Microsoft, Seattle, CA, USA).
3 Methods
The procedures below describe techniques to label paraffin-embedded histological material. The methods outline preparation of sections, the use of immunofluorescent staining to localize intracellular HDAC expression in situ, and finally, a quantitative analysis of immunofluorescent intensity to examine changes in HDAC expression.
3.1 Tissue Fixation, Embedding, and Sectioning
1.
Immerse in cold freshly prepared 4 % PFA, 10× the volume of tissue.
2.
Store at −4 °C for 2–18 h, depending on size of tissue.
3.
Wash in 0.01 M PBS for 10 min.
4.
Dehydrate tissue through graded alcohols (50, 70, 95, 100 %) and clear in two changes of chloroform (45 min to 1 h each).
5.
Infiltrate tissue with two changes of paraffin wax (approx 56 °C) for a total of 4–6 h. Excess heat should be avoided as it may potentially affect antigenicity.
6.
Orientate and embed tissue in fresh wax using molds and embedding cassettes.
7.
Place tissue and molds in a −20 °C freezer for a minimum of 2 h before separating the block from the mold.
8.
Heat water bath to 56 °C.
9.
Prepare a flotation bath by filling a small bowl with 20 % v/v ethanol in water.
10.
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Cut 2–5 μm sections of paraffin-embedded tissue with a microtome, in accordance with the manufacturer’s instructions.
Related posts:
Propagation and Culture of Human Renal Fibroblasts
Isolation and Primary Culture of Murine Podocytes with Proven Origin
Isolation and Propagation of Rat Peritoneal Mesothelial Cells
An In Vitro Murine Model of Vascular Smooth Muscle Cell Mineralization
The Isolation and Quantitation of Fetuin-A-Containing Calciprotein Particles from Biological Fluids
Intravital Multiphoton Imaging of the Kidney: Tubular Structure and Metabolism
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