high-fat diet plus streptozotocin-induced type 2 diabetes C57BL/6J mouse

FFPE 肾组织切片的 PAS 染色流程，按源方案 FFPE 分支执行脱蜡、复水、氧化、Schiff 显色、苏木素复染、脱水透明和封片。具体切片厚度、视野与定量方法按研究设计确定。

340 nm吸光度变化对应的葡萄糖浓度。

输出为达到既定高血糖阈值的成模动物；本方案不主张与某一原论文的给药操作等同，仅为可采用的诱导实现。

肾小球基底膜、系膜基质和糖蛋白呈 PAS 阳性（品红/紫红色），细胞核呈蓝色；按研究设计进行肾小球系膜扩张或病变范围判读。

输出为每样品尿白蛋白浓度（mg/l），按标准曲线插值报告；该读数本身不是白蛋白/肌酐比值，换算比值需另测尿肌酐。

Calibrate Cobas for Glucose analysis by running a multi analyte standard and two control serum. Calibrate Cobas for Glucose analysis by running a multi analyte standard and two control serum. Sample Handling as performed by the Cobas Mira Plus. Sample Handling as performed by the Cobas Mira Plus. a) Pipette 3 µL of sample into cuvette. b) Absorbance is measured at 340 nm. c) Add 100 µL of Glucose liquid reagent. d) Mixture is incubated at 37ºC for 10 minutes. e) Absorbance is measured at 340 nm. Change in absorbance is calculated.

Prepare the buffers and solutions as described below. Please note that the STZNa-Citrate solution should be prepared imm Prepare the buffers and solutions as described below. Please note that the STZNa-Citrate solution should be prepared immediately prior to injection so as to avoid degradation of the STZ. Mice should be fasted prior to injection; four hours is usually sufficient. Mice should be fasted prior to injection; four hours is usually sufficient. Place one mouse in the Isoflurane drop jar, according to your local IACUC anesthesia standards. Place one mouse in the Isoflurane drop jar, according to your local IACUC anesthesia standards. Remove mouse when breathing has slowed and animal is anesthetized Remove mouse when breathing has slowed and animal is anesthetized Inject appropriate amount of the STZ solution IP so the final dosage is **50 mg/kg mouse** Inject appropriate amount of the STZ solution IP so the final dosage is **50 mg/kg mouse** Allow mouse to awaken and place back in cage Allow mouse to awaken and place back in cage Repeat procedure for each animal; keep in mind that Isoflurane will need to be reloaded after every third or fourth anim Repeat procedure for each animal; keep in mind that Isoflurane will need to be reloaded after every third or fourth animal for proper anesthesia. Each mouse should be given one injection for 5 days consecutively. Each mouse should be given one injection for 5 days consecutively. Supply mice with 10% sucrose water, if necessary, to avoid sudden hypoglycemia post-injection. Supply mice with 10% sucrose water, if necessary, to avoid sudden hypoglycemia post-injection. **Mice should be tested for sufficient levels of hyperglycemia at 4 weeks postinjection. **If any of the experimental an **Mice should be tested for sufficient levels of hyperglycemia at 4 weeks postinjection. **If any of the experimental animals are not sufficiently diabetic, the low dose STZ protocol should be re-administered to those mice only at week 7 post injection to obtain proper levels of hyperglycemia.

Start with FFPE here: Allow PAS “kit” to come to room temperature on the bench. Start with FFPE here: For paraffin sections, deparaffinize in xylene, two changes, 00:03:00 each. Start with FFPE here: Hydrate through graded alcohols, 00:01:00 each: 100%, 100%, 95%, 70%, water Start with FFPE here: Rinse well in distilled water by holding finger over slides, pouring water into sink and adding water. Do this 5 times. Start Frozen samples here: Place in 0.5% Periodic acid for 00:10:00 (to oxidize). Start Frozen samples here: Rinse well in distilled water (follow Step 4). Start Frozen samples here: Pour Schiff’s reagent into Coplin jar containing slides. Allow to sit 15-30 minutes at room temperature (kidney samples ~30 minutes). Start Frozen samples here: Rinse in running warm (~40 °C) distilled or tap water (follow Step 4). The tissue should appear pink after rinsing with warm water. Start Frozen samples here: Place slides in movable gray slide holder. Start Frozen samples here: Counterstain in Hematoxylin (staining line) for 00:01:00. Start Frozen samples here: Rinse well in distilled water, starting with blue container next to hematoxylin (follow Step 4). Start Frozen samples here: Quickly dip slides into “Bluing” agent (0.5% ammonium hydroxide). Start Frozen samples here: Rinse 1 minute in distilled water until pink color is visible (follow Step 4). Start Frozen samples here: Dehydrate through graded alcohols, 10 short dips in each: 95%, 95%, 100%, 100% If the last 100% ethanol rinse is colored by residual stain, additional washing in new 100% ethanol solution should be performed. Start Frozen samples here: Fix in 2 rounds of xylenes, 00:01:00 each (in the hood). Start Frozen samples here: Coverslip slides: - Place coverslip on paper towel - Add 2-3 drops of cytoseal to edge (depending on size) - Dip slide into xylenes, take out and roll the xylene lengthwise on slide - Line up slide and cover slip and slowly place the slide on the coverslip - If needed, use dissecting tool to remove bubbles Start Frozen samples here: Image slides using a Leica brightfield scanner, AxioScan Z1 slide scanner (or equivalent) at 20x (~1 µm spatial resolution) and save as.tiff or.jpg file.

Prepare working reagent. Prepare working reagent. Prepare standards by serially diluting 200 mg/l standard 1:1 to make 100, 50, 25, 12.5, 6.25 standards. Prepare standards by serially diluting 200 mg/l standard 1:1 to make 100, 50, 25, 12.5, 6.25 standards. Add 25 μl of standard and sample to each well. Add 25 μl of standard and sample to each well. Add 125 μl of working reagent. Read in fluorimeter using 590 nm excitation and 616 nm emission. Add 125 μl of working reagent. Read in fluorimeter using 590 nm excitation and 616 nm emission. _**IMPORTANT: **__Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader._ Use a polynomial 2nd order curve fit to construct a standard curve. Interpolate the values of the unknowns using the sta Use a polynomial 2nd order curve fit to construct a standard curve. Interpolate the values of the unknowns using the standard curve.