But beyond that, the sections on Pakistan are a mixture of potted history and justifications of her reign and that of her father.
After that, the sections were incubated for 1 h with the subsequent primary monoclonal antibody.
After that, the sections were air-dried for 30 60 min. Laser microdissection was performed on a Leica ASLMD laser capture microdissection system (Leica Microsystems, Wetzlar, Germany).
After that, the sections were stained with a solution of 1% uranyl acetate in 70% ethanol for 1 hour and further dehydrated in ethanol.
After that, the sections were stained with 3, 3´-diaminobenzidine solution and counterstained with hematoxylin solution.
After that, the sections were washed with PBS and sealed with a coverslip.
After that, the sections were postfixed in 1% paraformaldehyde for 10 min.
Following that, the sections were incubated for 10 min with diaminobenzidine (DAB; DakoCytomation) to visualize the TUNEL-positive cell nuclei.
After that, the sections for HBME-1 and CD68 were incubated with the Envision+ polymer reagent (Dako, Japan).
After that, the sections were rinsed with PBS and treated with normal blocking serum (Vector Laboratories, Inc., Burlingame, CA, USA) for 30 min.
After that, the sections were washed in PBS and treated using the Novolink Polymer Detection System (Novocastra, Newcastle upon Tyne, UK) according to the manufacturer's instructions.
