BioBreeding (BB) rat

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。

血糖浓度读数。

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。

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.

**Measurement of Blood Glucose ** **Measurement of Blood Glucose ** Blood samples were collected from the tail vein for the determination of blood glucose levels using Accutrend Plus test strips and meter (Roche).

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.