3.1 Immunohistochemistry for Pten, p-Akt and p-4EBP1 in FFPE tissue

For cryosections, mice were anesthetized and perfused transcardially with PBS followed by 4% paraformaldehyde (PFA) in PBS. Following dissection, tissues were post-fixed overnight in 4% PFA in PBS at 4°C, and then equilibrated in 25% sucrose in PBS for an additional 24 hours at 4°C. Tissues were embedded in embedding media OCT (Triangle Biomedical Sciences) on dry ice and cut into 12 μm-thick cryosections. Tissue slides were equilibrated at room temperature for 20 minutes then washed three times in PBS prior to staining.

For paraffin sections, tissue was processed the same way as above except after dissection, tissue was postfixed for 24 hours in 4% PFA in PBS at 4°C, then processed and embedded in paraffin, and cut into 5 μm sections.

For optimal IHC, tissue should not be left in fixing solution for more than 4 days before being processed for paraffin-embedding or equilibrated in 25% sucrose in PBS for cryoprotection followed by embedding in OCT for cryosections.

Transfer slides through the following series of solutions (See ^{Note 1} and ²):

Xylenes 5 – 10 min

Xylenes 3 min X 2

100% EtOH, 2 min X 2

95% EtOH, 2 min

70% EtOH, 1 min

50% EtOH, 1 min

20% EtOH, 1 min

8. H₂O, 2 min X 2

Saturation: Place sections in plastic Coplin jar(s) filled with the antigen-retrieval solution completely to the top as below:

For deparaffinized sections: antigen-retrieval solution 10 min

Microwave. Prepare a total of 4 plastic Coplin jars either with or without slides (jars without slides can be filled with H₂O)

Seal the jar(s) as tightly as possible and position them at the center of a microwave oven with rotating tray

After each microwave interval, refill Coplin jars if any solution has leaked out.

2.5 min at power 100%

2.5 min at power 50%

2.5 min at power 50%

2.5 min at power 50%

Open the lid and cool the solution to room temperature (at least 30 min; See ^{Note 3})

Wash sections with TBS in Coplin jars (up to 13 slides/jar) on belly dancer, 10 min

Treat sections with freshly made 1% H₂O₂ (in TBS) for 30 min at room temperature

Wash with TBS, 5 min X 2, and with TBS-T, 5 min X 1, in Coplin jars on belly dancer

After the second wash draw a boundary with a wax pen around the area of the slide to be stained (See ^{Note 4})

Block non-specific binding sites with 10% goat serum (in TBS-T) for 30 min to 1 hr, usually 300~500 ul for each slide in a humidified chamber at room temperature.

Dilute primary antibody in 2% goat serum (in TBS-T). (~300uL/full slide) in a humidified chamber at room temperature. A. anti-Pten: 1:100 dilution. B. anti-p-Akt antibody 1:50 dilution. C. anti-p-4EBP1 1:500 dilution

Remove the blocking solution and incubate the sections with the primary antibody in a humidified chamber overnight at 4°C

Wash the sections with TBS-T in Coplin jars on belly dancer, 5 min X 3. (See ^{Note 5}). Incubate the sections with biotinylated anti-rabbit IgG antibody diluted to 1:200 in 2% goat serum (in TBS-T) for 1 hr in a humidified chamber at room temperature

Prepare Elite ABC solution at least 30 min before use: To 2.5 ml of TBS-T, add 2 drops of solution A. Mix by vortexing and add 2 drops of solution B.

Mix by vortexing and leave the solution at room temperature

Wash the sections with TBS-T, 5 min X 2, and 10 min X 1, in a humidified chamber at room temperature

Optional tyramide amplification of p-Akt signal: (i) Wash the sections with TBS-T, 5 min x 3. (ii) Add the biotinylated-Tyramide (1:175 dilution in TBS-T) and incubate for 10 min at room temperature (300~500 ul/slide). (iii) Wash the sections with TBS-T, 5 min x 3. (See ^{Note 6}).

Add the ABC solution and incubate for 1 hr in a humidified chamber at room temperature (300~500 ul/slide), or for 30 minutes if tyramide amplification was used. (See ^{Note 7} and ⁸).

IHC signals are visualized by incubating with NovaRED or diaminobenzidine (DAB), HRP substrates that produce a red or dark brown reaction product, respectively. NovaRED is generally more sensitive than DAB. The optimal substrate depends on the level of signal associated with the phenotype.

Wash with TBS-T, 5 min X 2, and 10 min X 1.

During the last wash prepare NovaRED solution (or DAB solution). To 5 ml of water, add 3 drop of reagent 1 and mix well by vortexing. Add 2 drops of reagent 2 and mix by vortexing. Add 2 drops of reagent 3 and mix by vortexing. Add 2 drops of H₂O₂ reagent and mix by vortexing.

Apply the solution onto washed sections. Incubate until signals become distinguishable between sample and control. Pten usually takes approximately 7–10 min. p-Akt takes approximately 10–15 min without Tyramide amplification and 4 min with Tyramide amplification. p-4EBP1 takes approximately 10 min.

Optimal incubation time will vary with tissue preparation. Also, if detecting pathological activation of the pathway, the optimal detection time will depend on the level of pathway activation. Observe under the microscope to select the optimal time to stop the development reaction. Representative immunohistochemistry images are shown in Figure 1.

A-F: Nestin-creER; Pten^flox/flox with cre activity induced by tamoxifen administration at postnatal days 0 and 1, with tissue collected at day 30. A-C: Pten^wt mouse; D-F: Pten^ko. G-H: Nestin-creER; Pten^flox/flox;Tp53^flox/flox mouse medulloblastoma. I: GFAPcreER; Pten^flox/flox;Tp53^flox/flox mouse astrocytoma.

(A and D): Pten IHC: mouse dentate gyrus, Pten wild-type cells are red while Pten-null cells in the inner layer of the dentate gyrus stain only with hematoxylin counter stain. (B and E): p-Akt IHC,: Pten-null dentate gyrus neurons are strongly positive (red) for p-Akt. (C and F): p-S6 IHC: Pten-null dentate gyrus cells are more strongly positive (red) for p-S6 than the baseline p-S6 level in wild-type mice. G. Pten IHC, mouse medulloblastoma: Pten-null tumor cells stain only with hematoxylin counter stain while wild-type blood vessel endothelial cells are brown. H. p- Akt IHC, mouse medulloblastoma, Pten-null tumor cells are strongly positive (red) while the adjacent normal brain is negative for p-Akt. I. p-4ebp1 IHC, GFAP-creER;Pten;Tp53 double knockout mouse astrocytoma are strongly positive for p-4ebp1. Scale bar = 50 μm. Substrate: Nova red, counterstain, hematoxylin.

Discard the solution into a beaker and stop the development by washing sections with water 5 min X 2. (See ^{Note 9})

Incubate the sections in hematoxylin QS solution for 15 seconds.

Rinse the sections in a Coplin jar with running tap-water until the rinse is colorless (5–10 min).

Dip the slides in water 10 times

Dehydration: 20% EtOH 1 min, 50% 1 min, 70% 1 min, 95% 2 min, 100% 2 min X 2, Xylenes 5 min X 3

Coverslip with Permount in the fume hood

Leave mounted slides in the hood at least overnight to dry.