Immunostaining

Phase-contrast observation of mutant testes can only give a broad indication of the cytological defect in any particular mutant. Immunostaining mutant testes can give information on both cell-type distributions and subcellular structures. At the subcellular level, for example, staining of the meiotic spindle with anti-tubulin antibodies and a DNA dye will reveal much about the cytological mechanism underlying a chromosome nondisjunction phenotype. Antibodies or markers can also reveal which cell types are present in the testes. This is especially important in situations where relatively few, or very small, cells are seen in phase contrast. Are these germ-line or somatic cells? Are they stem cells or spermatogonia? Is the hub present and normal? Many of these cell type-specific markers are LacZ enhancer traps, so staining can be done either with an anti-LacZ antibody (Subheading 3.2.) or with a P-galactosidase activity assay (see Subheading 3.3.). A selection of useful antibodies and markers for probing subcellular structure and cellular identity in the testis is given in Tables 3 and 4. The detailed analysis of mutant phenotypes is, of course, not the only reason for wanting to immunostain testes. Having generated an antibody to a protein expressed in testes, one will always want to know its cellular and subcellular distribution patterns.

The choice of the following protocols depends on the exact question being addressed in the experiment. If information is needed on the pattern of protein localization at a global scale, then clearly whole-mount immunohistochemis-try is required. This has the advantage of maintaining the temporal pattern of cysts in the testes; however, the resolution at the subcellular level is relatively low. This can be somewhat helped by counterstaining the preparation with Hoechst 33258. Immunofluorescence of squashed preparations has greater resolution of subcellular structures, but at the cost of loss of the temporal

Table 3

Choosing Antibodies and Reagents for Labeling

Table 3

Choosing Antibodies and Reagents for Labeling

Antibody/probe

Structure revealed

Suggested conc.

Supplier (ref.)

DAPI

DNA

1 |g/mL

Molecular Probes

Propidium iodide

DNA

1 |g/mL

Sigma

(see Figs. 2 and 3)

Hoechst 33342

DNA (vital dye)

2-5 |g/mL

Sigma (34)

(see Fig. 1)

Phospho-Histone H3

Mitotic/meiotic

1:200

Upstate Biotechnology (36)

(see Fig. 3)

chromosomes

Phalloidin

F-actin Hub

5 |g/mL

Sigma/Molecular Probes (27)

Cleavage furrow Investment cone Muscle of sheath

Tubulin (YL1/2)

Microtubules

1:20 (tissue culture supernatant)

Serolab (14)

Fasiclin III

Hub

1:50

(43)

Phospho-tyrosine

Ring canals

1:100

Sigma/Upstate Biotechnology (26)

Spectrin

Fusome

1:50

(44,45)

mAblbl (adducin)

Bam-C

Spermatogonia

1:1500

(46)

Anillin

Nuclei and ring canals

1:300

(26)

Gamma-tubulin

Centrosomes

1:400

(47)

MPM2

DNA spindles and centrosomes

Upstate Biotechnology (14)

ß-galactosidase

LacZ reporter patterns

Enhancer Traps That Reveal Specific Cell Types and GFP Reporters for Substructures

Reporter line Cell type/structure Ref.

M34a Germ-line stem cells and gonialblasts 36

LacZ600, eyes absent Hub and cyst cells 37,48

Vein Cyst cells 36

M5-4 Hub, germ-line stem cells and gonialblasts 49

Don Juan GFP Sperm tails 50

His2-GFP Meiotic chromosomes 41

sequence of cysts. Two different fixation protocols for immunofluorescence of squashed preparations are given here. The choice of which to use is partly personal preference and partly antibody/antigen-specific. As a general rule, if trying a new antibody, do both protocols. I find that the methanol/acetone method (Subheading 3.2.3.) gives better fixation of meiotic spindles. However, phalloidin staining of F-actin is significantly better with formaldehyde fixation (Subheading 3.2.2.).

3.2.1. Whole Mount Immunohistochemistry

1. Dissect the testes in the appropriate buffer and transfer to an Eppendorf tube (see Notes 2 and 6).

2. Fix the testes in 4% formaldehyde in PBS, for 20-30 min at room temperature.

3. Rinse once, transfer to tissue culture inserts in a 24-well tissue culture plate, then wash three times in PBSTx, 20 min each wash (see Note 7).

5. Incubate in 1° antibody diluted in PBSTx + 5% FCS overnight at 4°C, or at room temperature for 2 h (see Note 9).

6. Rinse once, then wash three times in PBSTx, 20 min each.

7. Incubate in biotinylated 2° antibody diluted 1 : 2000 in PBSTx + 5% FCS for 1 h at room temperature.

8. Rinse once, then wash three times in PBSTx, 20 min each. (If using Vectastain ABC kit, see Note 10.)

9. Incubate for 30 min at room temperature with ExtrAvidin-HRP conjugate diluted 1 : 1000 in PBSTx.

10. Rinse once, then wash three times in PBSTx, 20 min each.

11. Stain with 0.7 mg/mL DAB, 0.001% H2O2 in PBS (see Note 11).

12. Stop the reaction by rinsing once, then washing several times (10 min each) in PBS.

13. Counterstain DNA with 1 |g/mL Hoechst 33258 in PBS for 15 min if required.

14. Mount on slides in mounting medium and observe with Nomarski optics (see Note 12) (see Fig. 8).

3.2.2. Formaldehyde Fixation for Confocal Microscopy

1. Dissect testes from young adults in testis buffer, four or five pairs per slide (see Note 2).

2. Transfer the testes to a drop of testis buffer on a poly-L-lysine-treated slide and cut open to spill the contents (see Notes 3 and 13).

3. Squash gently under a 22 x 22-mm2 siliconized cover slip.

4. Freeze immediately in liquid nitrogen (wear safety glasses) and pop off the cover slip using a scalpel.

5. Immediately place in chilled (on dry ice) 100% ethanol, 10 min.

6. Place the slide flat and flood with 0.5-1 mL of 4% formaldehyde solution; incubate for 7 min.

7. Tip off the formaldehyde onto paper towels (wear gloves). If staining with phal-loidin, see Note 14.

8. Permeabilize the testes by incubating twice in PBST-DOC, 15 min each.

9. Store in PBST until all the slides have been prepared.

10. Make a well on the slide with Evostick (rubber cement), block for a minimum of 30 min in PBST + 3% BSA or PBST + 5% FCS (see Note 15).

11. Incubate with primary antibody diluted in PBST + BSA (or PBST + FCS) for a minimum of 2 h at room temperature, or overnight at 4°C in a humid chamber (see Notes 9 and 16).

12. Remove the evostick. Wash 4 times, 15 min each, in PBST.

13. Incubate with secondary antibody 1 : 500-1 : 1000 (in fresh Evostick wells) for at least 1 h at room temperature, or overnight at 4°C, in a humid chamber (see Note 16).

14. Remove the Evostick. Wash four times in PBST, 15 min each.

15. Optional: Incubate 10 min in 1 |g/mL DAPI in PBST.

16. Wash once in PBS for 10 min.

17. Mount under a siliconized cover slip in 85% glycerol + 2.5% «-propyl gallate (containing 1 |g/mL propidium iodide if required). Seal with nail polish.

18. Image with conventional epifluorescence or confocal laser scanning microscopy.

3.2.3. Methanol/Acetone Fixation for Confocal Microscopy

1. Dissect testes from young adults in testis buffer or TB1, four or five pairs per slide (see Note 2).

2. Transfer to a drop of testis buffer on a poly-L-lysine-treated slide and cut open to spill the contents (see Note 3).

3. Squash gently under a 22 x 22-mm2 siliconized cover slip.

4. Freeze immediately in liquid nitrogen (wear safety glasses) and pop off cover slip using a scalpel.

5. Transfer to methanol, chilled on dry ice, and incubate for 5 min.

6. Transfer to acetone, chilled on dry ice, and incubate for 5 min.

7. Incubate in PBS-T-AA for 10 min at room temperature.

8. Store in PBST until all the slides are prepared.

9. Continue from step 10 of Subheading 3.3.2.

Fig. 8. Immunohistochemistry of Aly and dpERK protein localization. Immunohis-tochemical staining retains the spatial relationship between different stages of differentiation in the testes, and so allows temporal changes in the cellular or subcellular distribution of an antigen to be assessed. (A) The tip of a testis stained with an antibody against Aly protein. Aly is not expressed in the mitotic domain (out of focus in this image). When Aly is first detected, it is both cytoplasmic and nuclear in the germline cells. As the cysts mature, the localization resolves to be concentrated on chroma-tin in primary spermatocytes. Aly protein is not found in cyst cells; hence, the gaps seen between the areas of staining. (B) A monoclonal antibody specific to the activated (diphosphorylated) form of ERK/MAP kinase stains only the cyst cells near the apical tip of the testis. This staining is particularly sensitive to buffer conditions and only works when testes are dissected in TBPi buffer.

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