Immunohistochemical Staining Steps 40

Immunohistochemical Staining Steps [40]

2.4.1 After dewaxing and hydration of the paraffin sections, rinse three times with PBS solution (pH 7.5 ± 0.1), 3 minutes each time.

2.4.2 According to the requirements of the monoclonal antibodies, add 1 ml of EDTA repair solution (pH 8.0) to repair the tissue antigens.

2.4.3 Add one drop of peroxidase-blocking solution to each section, incubate at room temperature for 10 minutes to block the activity of endogenous peroxidase, then rinse three times with PBS solution, 3 minutes each time.

2.4.4 Remove the PBS solution, add one drop of normal non-immune animal serum to each section, incubate at room temperature for 10 minutes.

2.4.5 Remove the animal serum, add the primary antibody to each section, incubate at room temperature for 60 minutes.

2.4.6 Rinse three times with PBS solution, 5 minutes each time, remove the PBS solution, add one drop of biotin-labeled secondary antibody KT-9707-c, Biotin-conjugated second Antibody (Rabbit), incubate at room temperature for 10 minutes, then rinse three times with PBS solution, 3 minutes each time.

2.4.7 Remove the PBS solution, add two drops of freshly prepared DAB solution to each section, observe under the microscope for 3–10 minutes.

2.4.8 Rinse with tap water, counterstain with hematoxylin, then rinse again with PBS solution to restore the blue color.

2.4.9 DAB staining, all sections undergo gradual dehydration using alcohol at different concentrations, followed by xylene clearing and neutral gum sealing.

Using the peroxidase–anti-peroxidase method, the final reaction product appears brown, and after processing with image analysis software, the percentage of positive cells is determined based on the average optical density value and the target area ratio.

Table 4: Effects of Pei's Blood-Elevating Granules on T-Lymphocyte Subpopulation Content in Aplastic Anemia Mice (x̄ ± S)

Note: Compared with the model group, #p < 0.05, *p < 0.01; compared with the normal group, △p < 0.05, ○p < 0.01

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Figure 8: Effects of Pei's Blood-Elevating Granules on T-Lymphocyte Subpopulations in Aplastic Anemia Mice

As shown in Figure 8, compared with the normal group, the aplastic anemia model group exhibits a significant decrease in CD4 values and a significant increase in CD8 values, resulting in a lower CD4/CD8 ratio. After 40 days of medication, the medium- and low-dose groups of Pei's Blood-Elevating Granules show a significant increase in CD4 values and a significant decrease in CD8 values, while the high-dose group and the Zhenqi group do not have a significant effect on increasing CD4 or decreasing CD8. The CD4/CD8 ratio only increases slightly, with no significant difference compared with the model group. Analysis: A large body of research has demonstrated that patients with aplastic anemia exhibit an imbalance in T lymphocyte subsets, with abnormal activation of CD8 cells, leading to excessive secretion of suppressive cytokines such as IL-2, IFN-γ, and TNF-α [41]. These cytokines synergistically exert negative regulatory effects on hematopoiesis, resulting in defects in the proliferation and differentiation of hematopoietic stem and/or progenitor cells [42]. The mechanism of action of Pei's Shengxue Granules may be multifaceted: on one hand, it can regulate the body's dysregulated immune function, increase the CD4/CD8 ratio, reduce the number and activity of CD8 cells, thereby decreasing the release of hematopoiesis-suppressive factors such as IL-2 and IFN-γ and relieving the inhibition of bone marrow hematopoiesis [43]. Cytokines such as IL-2, TNF-α, and IFN-γ not only directly inhibit the proliferation of hematopoietic cells [44], but also induce programmed cell death of CD34 cells through Fas antigen (CD95)-mediated apoptosis, ultimately leading to hematopoietic failure [45]. In addition, these cytokines can directly promote the proliferation and differentiation of hematopoietic stem/progenitor cells, particularly erythroid hematopoiesis. Immunohistochemical detection of CD4 and CD8 reveals that CD4 and CD8 in aplastic anemia patients are in an early activated state [46]. The above reagent kits were purchased from Changsha Yingrun Biological Products Co., Ltd., batch number: GT200310, and all operations were performed according to the kit instructions.

3 Statistical Analysis

Experimental results were analyzed using SPSS 10.0 statistical software, with all data expressed as mean ± standard deviation (x̄±s). Student's t-test was used for comparison between two groups, while one-way ANOVA was employed for comparisons among multiple groups; p<0.05 was considered statistically significant.

Discussion

Pei's Shengxue Granules is one of the well-known anti-tumor and immune-enhancing prescriptions developed by Professor Pei Zhengxue, a nationally renowned expert in integrated traditional Chinese and Western medicine, based on his many years of clinical experience. It is a pure herbal granular preparation with effects such as tonifying the kidney and generating marrow, strengthening the spleen and benefiting qi, nourishing blood and harmonizing the stomach. Clinical application studies of this formula were conducted in 2002 as a scientific research project of Gansu Province and subsequently passed the assessment of scientific and technological achievements [38].

Research on Pei Zhengxue’s series of formulas

This achievement was awarded the First Prize of the Huangfu Mi Science and Technology Achievement Award in Traditional Chinese Medicine of Gansu Province in 2003. Due to its success in curing multiple cases of leukemia, the formula was named the “Lanzhou Formula” at the National Hematology Conference in 1973 and has been clinically applied for over 30 years. In 1997, it was formulated as an internal preparation of the Gansu Provincial Academy of Medical Sciences under the name “Pei's Shengxue Granules.” Years of clinical practice have proven that this formula exhibits remarkable clinical efficacy in treating immune system dysfunction and malignant tumors, especially immune deficiency caused by radiotherapy and chemotherapy in cancer patients. To date, it has been used to treat tens of thousands of patients both within and outside the province, with its clinical effectiveness recognized by clinicians and patients alike, earning it considerable fame domestically and internationally.

1 Indicator Selection