3 3 diaminobenzidine dab

Lithium chloride, pilocarpine, diazepam, scopolamine, and MK-801 were purchased from Sigma. Propofol was purchased from AstraZeneca. Rabbit polyclonal antibodies against mouse and human GABA_A receptor a1 subunits were purchased from Alomone. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and 3,3-Diaminobenzidine (DAB) were purchased from ZSBIO. Protein BCA assay kit, Western Blot Stripping Buffer, skimmed milk powder, nitrocellulose membrane, and Super ECL Plus were purchased from Beijing Applygen Technologies. Wide-range prestained protein marker (molecular weight standard: 6–200 kd) was purchased from New England Biolabs. Other assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute.

Paraffin blocks of tumor were sliced into 4 μm-thick sections and dried onto slides. The sections were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide for 30 min. For antigen retrieval, slides were immersed in citrate solution (pH 6.0) heated at 121 °C for 20 min, and then blocked with 3% normal horse serum for 30 min. The slides were incubated with anti-Ki-67 antibody (1:400) or anti-cleaved caspase-3 antibody (1:100) overnight at 4 °C followed by incubated with HRP-conjugated anti-rabbit IgG antibody (ZSGB-BIO, Beijing, China) for 1 h. The immunoreactivities were detected with 3,3′-diaminobenzidine (DAB, ZSGB-BIO) and examined under an Eclipse Ci microscope (Nikon, Japan).

Expressions of fibronectin, collagen I, α-SMA, and E-cadherin in the kidney tissues of mice were determined by immunohistochemistry. Paraffin-embedded kidney tissue sections were deparaffinized, rehydrated, and subjected to microwave-based antigen retrieval in citrate buffer. A blocking step was performed using 10% normal goat serum. The kidney sections were incubated overnight at 4 °C with anti-fibronectin, collagen I, α-SMA, and E-cadherin primary antibodies, and with polyperoxidase-anti-mouse IgG (ZSGB-BIO, Beijng, China). Immunoreactive signals were developed upon incubation with 3,3-diaminobenzidine (DAB, ZSGB-BIO). Images of fibronectin, collagen I, α-SMA, and E-cadherin were obtained and photographed under a microscope (Olympus Corporation, Tokyo, Japan) at 200× magnification.

DA5‐CH had been synthesized by China Peptides. It had a purity of 95%. Its purity was confirmed by reversed‐phase high‐performance liquid chromatography (HPLC) and characterized by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. The drug was stored at −20°C and dissolved in saline before intraperitoneal injection in rats. Streptozotocin (STZ) was bought from Sigma‐Aldrich. It was stored at −20°C and dissolved in artificial cerebrospinal fluid before lateral ventricular injection in rats.
Rabbit anti‐tau, p‐tau (phospho ser396), synaptophysin, p‐CREB (phospho Ser133), CREB, and anti‐rabbit IgG were purchased from Abcam. Antibodies against B‐celllymphoma‐2 (Bcl‐2), Bax were bought from Boster. The antibody to PSD95 was obtained from Proteintech. BCA protein assay kit and Mayer's Hematoxylin solution were purchased from Solarbio. Sodium chloride, ethylene glycol, and 3,3‐diaminobenzidine (DAB) were obtained from ZSGB‐BIO Co.

The upper and middle lobes of right lung were fixed in 10% formalin for 24 h. The tissues were dehydrated, embedded in paraffin and cut into 5 mm sections. The tissues were stained with hematoxylin and eosin (HE staining) after deparaffinization, and evaluated under an optical microscopy (Olympus BX51, Japan).
The expression of ICAM-1 in lung was determined by immunostaining. After deparaffinization and rehydrating, paraffin sections were placed into a pressure cooker containing antigen retrieval buffer (0.01 M citrate buffer, pH 6.0), cooked with full pressure for 2 minutes to unmask antigens. Immunostaining was performed by incubating the sections with mouse anti-rat ICAM-1 monoclonal antibody (1∶200, Abcam, MA) overnight at 4°C, biotin-conjugated secondary antibody (ZSGB-bio, China) at 37°C for 1 h, and streptavidin-HRP (ZSGB-bio, China) at 37°C for 30 min. 3,3-Diaminobenzidine (DAB, ZSGB-bio, China) was then used to visualize immunohistochemical staining. Cell nuclei were counterstained with hematoxylin. Images were obtained with an Olympus BX51 microscope and the proportion of positive staining cells was analyzed with Image-Pro plus 5.1 software. The expression of ICAM-1 in lung tissue was presented as mean photodensity.
For histology and immunostaining analysis, the slides were renamed by arabic number followed by a double-blinded examination by two pathologists.