Development and Validation of a Methodology for Establishing Obese Rat Models with Typical Fatty Pancreas

Summary

This study offers a reproducible and standardized method in order to establish obese rat models that exhibit simple obesity, commonly characterized by fatty pancreas. It also includes detailed protocols for pancreatic tissue sampling and immunohistochemistry.

Abstract

Obesity has emerged as a predominant global health epidemic, with nearly half of the world's population now classified as overweight or obese. A key pathological feature in obese individuals is ectopic lipid deposition in non-adipose tissues, including the pancreas-a condition termed pancreatic steatosis or fatty pancreas. In high-fat diet (HFD)-induced rodent models, pancreatic steatosis consistently precedes hepatic steatosis, underscoring the particular susceptibility of the pancreas to lipid accumulation. This early involvement positions the pancreas as a critical organ for understanding metabolic dysregulation in obesity. Although diet-induced obese (DIO) rats recapitulate core aspects of human disease progression, well-characterized animal models that reliably exhibit this pancreatic phenotype remain scarce in the literature.

A significant methodological consideration in modeling human obesity is the timing of HFD introduction. Many existing models initiate HFD during the weaning period, which may introduce confounding developmental metabolic programming effects that do not fully reflect human obesity, which primarily develops post-developmentally. In contrast, the present study establishes a standardized protocol in which HFD is introduced post-weaning. This approach more accurately mimics the common human trajectory of obesity onset in adulthood, avoids early-life metabolic adaptations, and results in a more physiologically relevant progression toward pancreatic steatosis.

This research provides a detailed and reproducible framework for generating a rat model of obesity characterized by prominent fatty pancreas pathology. The methodology includes a 1-week acclimatization phase, a 14-week HFD induction period starting after weaning, and systematic tissue harvesting. Two tissue processing pathways are described: snap-freezing for molecular analyses (e.g., Western blotting) and paraformaldehyde perfusion-fixation for morphological evaluations (e.g., IHC, H&E). This model offers a robust platform for mechanistic investigations into obesity-associated pancreatic metabolic dysfunction.

Introduction

Simple obesity is defined as obesity that exists independently without other comorbidities, primarily attributed to energy imbalance resulting from unhealthy lifestyles, such as improper dietary patterns and insufficient physical activity¹. Obesity significantly contributes to the rising incidence of non-communicable diseases among younger populations, characterized by high prevalence rates, early onset, and multisystem complications, positioning it as a critical global public health challenge². Fatty pancreas is a condition marked by ectopic lipid deposition in this non-adipose organ during metabolic dysregulation. Fatty pancreas reflects a critical interplay between adipose tissue and pancreatic pathophysiology. Excessive pancreatic fat accumulation displaces normal acinar and islet tissues, inducing dual functional impairments in both exocrine and endocrine capacities^3,4. This pathological process disrupts α/β-cell ratios, promotes β-cell dedifferentiation with subsequent loss of insulin secretory function⁵, and mediates direct lipotoxic damage to β-cells⁶. Concurrently, α-cell proliferation and dysregulated insulin release exacerbate glycemic instability, accelerating progression to type 2 diabetes mellitus (T2DM) and its associated complications⁷.

Investigating fatty pancreas pathogenesis reveals novel mechanisms underlying metabolic disease progression⁸. While prior obesity research emphasized hepatic steatosis, emerging evidence identifies pancreatic lipid deposition as an earlier event in metabolic dysregulation⁹. This paradigm shift enables the construction of a dual-organ pathology model for simple obesity, integrating pancreatic and hepatic steatosis¹⁰. Studying their cross-talk may elucidate critical pathways across the disease spectrum spanning lipid metabolism disorder, obesity, insulin resistance, and diabetes, offering fresh insights into disease mechanisms¹¹.In the pathological process of the pancreas, the initial manifestation is significant deposition of pancreatic fat, accompanied by substantial expansion of pancreatic adipocytes. This is predominantly observed in the ventral aspect of the pancreatic head, neck, and body regions, and is primarily located within the interstitial areas between acinar and islet cells. Concurrently, chronic low-grade inflammation of the pancreas is present, while both endocrine and exocrine functions remain largely intact at this stage^12,13. Extensive fatty infiltration further impairs pancreatic β-cell function through lipotoxicity and suppresses insulin activity. During this phase, acinar cells are progressively replaced by adipocytes, thereby contributing to the development of T2DM^14,15. Although diabetes mellitus is not an independent risk factor for pancreatic cancer, patients with a history of acute pancreatitis and diabetes exhibit a significantly elevated risk of developing pancreatic cancer¹⁶.

Macroscopic anatomical analyses quantify pancreatic steatosis severity and infiltration patterns, while IHC evaluations at the microscopic level characterize acinar cell morphology, nuclear positioning, vacuolization frequency, and cellular population dynamics¹⁷. This multimodal approach, spanning gross anatomical observation to cellular-resolution IHC, enables comprehensive staging of pancreatic pathology across disease progression. The present study details a standardized protocol encompassing a 1-week acclimatization, a 14-week obesity induction in adult rats, and optimized pancreatic tissue processing for morphological analysis, establishing a robust framework for investigating steatosis-associated metabolic derangements.

Protocol

The animal treatment procedure was approved by the Animal Ethics Committee of Nanjing University of Traditional Chinese Medicine (202006A016) and complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines, ethical guidelines, guidance on the operation of the Animals (Scientific Procedures) Act 1986 and associated guidelines, EU Directive 2010/63 for the protection of animals used for scientific purposes. All experimental animals were anesthetized in accordance with approved rodent anesthesia procedures prior to dissection. The procedure was organized into three distinct components.

1. Establish an obese rat model

1. Experimental animals and groups
   1. Select male Sprague-Dawley (SD) rats aged 4 weeks (80-120 g) from a strain sensitive to high-fat diet (HFD).
   2. Acclimate all rats to a maintenance diet for one week, then randomly divide them into two groups: the control group and the model group.
   3. Replace the maintenance diet with HFD for the model group after one week, while maintaining the control group on the original diet. Maintain both groups on their diets for 14 weeks. The HFD composition is detailed in Table 1, and the standard maintenance feed formulation is in Table 2.
2. Feeding conditions and experimental parameters
   1. Environmental parameters: temperature 22 ± 2 °C, humidity 50 ± 10%, 12 h light/dark cycle, and free access to water and food.
3. Monitoring indicators
   1. Record body weight and food intake weekly at a fixed time.
      NOTE: Doing it in the evening can minimize stress and align with diurnal/nocturnal activity patterns¹⁸.
   2. Plot a body weight growth curve using the recorded data.
   3. Define successful obesity modeling as when body weight exceeds 20% of the age-matched control group.

Ingredient	Content (g/kg)	Major nutrients provided
Ordinary powdered feed	365	Basic nutritional supply of carbohydrates, Vegetable proteins, Fats, Vitamin complexes, Minerals, etc
Lard	310	Saturated fatty acids, Unsaturated fatty acids
Casein	250	Essential amino acids
Cholesterol	10	Cholesterol
Cellulose and mineral mixture	60	Fiber, Calcium, Phosphorus, Sodium, etc
DL-Methionine Methionine/Methionine Amino Acids	3	Essential amino acids
Yeast	1	B vitamins, β-glucan, etc
NaCl	1	NaCl

Table 1: Special HFD formulation for establishing DIO in rat models. The table details the specific composition of the HFD used in this study for the model group, which was administered from the end of the acclimation period until tissue collection.

Ingredient	Content	Unit
Moisture	12.196	%
Crude Protein (CP%)	18.272	%
Digestible Energy (DE)	3.186	Mcal/kg
Metabolizable Energy (ME)	2.939	Mcal/kg
Net Energy (NE)	2.228	Mcal/kg
Ether Extract (EE%)	4.417	%
Crude Fiber (CF%)	3.677	%
Calcium (Ca%)	0.991	%
Total Phosphorus	0.681	%
(Total P%)
Non-Phytate Phosphorus	0.19	%
Salt	0.407	%
Lysine	1.118	%
Digestible Lysine	0.813	%
Methionine	0.53	%
Threonine	0.779	%
Tryptophan	0.221	%
Copper (Cu)	22.06	mg/kg
Iron (Fe)	173.69	mg/kg
Manganese (Mn)	102.1176	mg/kg
Zinc (Zn)	60.3687	mg/kg

Table 2: Standard maintenance feed formula in rats. The table presents the formulation of the standard maintenance feed, which was provided to all rats during the acclimation phase and to the control group throughout the entire study duration.

2. Tissue harvesting

1. Preoperative preparation
   1. Fasting treatment: 12 h food restriction before euthanasia to minimize intestinal content interference with biochemical analyses.
   2. Anesthesia: Administer isoflurane via inhalation for deep anesthesia (induction concentration 4%, maintenance concentration 1.5-2%). The anesthesia machine was equipped with a vaporizer for isoflurane, and the anesthetic concentration was maintained at 1.5-2. As a reference for preparation, the physical consumption of liquid isoflurane was approximately 0.5 mL-0.8 mL per rat for the entire procedure.
   3. Monitor anesthesia depth: Continuously track respiratory rate and corneal reflexes. Maintain a sustained anesthetic state throughout the procedure to prevent postmortem pancreatic autolysis.
   4. Manage insufficient anesthesia: If anesthesia depth is inadequate or consciousness recurs, mmediately increase the concentration of isoflurane to supplement anesthesia and recheck vital signs before continuing.
      NOTE: Isoflurane anesthesia is the method compliant with the ethical requirements of the Experimental Animal Center of Nanjing University of Chinese Medicine. Additionally, the existing evidence suggests that isoflurane may offer a higher safety profile than ketamine in rodents¹⁹.
2. Fresh tissue harvesting
   1. Skin disinfection: Spray 75% ethanol rapidly over the entire body (1-2 s) to prevent hair contamination of abdominal organs.
   2. Position fixation: Position the rat supine on the dissection table and fix all four limbs securely with tape.
   3. Laparotomy: Grasp the abdominal skin with forceps, and using scissors, make a transverse incision along the lower edge of the costal margin. Use blunt dissection to separate the subcutaneous fascia and connective tissue.
   4. Pancreas identification: Gently displace the bowel to the left to visualize the stomach, spleen, and duodenum. The spleen is attached to the tail of the pancreas, and the stomach is close to the pancreas.
   5. Rapid dissection: Gently elevate the spleen with forceps and isolate the pancreas by progressively cutting its attachments to the spleen, stomach, and duodenal mesentery. Due to its fragile texture, avoid direct clamping of pancreatic parenchyma and preserve pancreatic ducts/blood vessels to prevent tissue tearing.
   6. Tissue preservation: Rinse the dissected pancreas rapidly with ice-cold PBS (1× Phosphate buffered saline, PH 7.4) or saline to remove residual blood. Immediately transfer the pancreas to a 2 mL freezing tube, submerge in liquid nitrogen within 30 s, and store long-term at -80 °C.
   7. Euthanasia and carcass disposal Following tissue collection, euthanize all experimental rats by cervical dislocation. According to the regulations of the Experimental Animal Center of Nanjing University of Chinese Medicine, animal carcasses are classified as pathological biomedical waste (Waste Code: 831-003-01). Seal all animal carcasses in airtight bags according to regulations, and store them uniformly in designated freezers. Centralized, environmentally sound disposal will be conducted by the university. Under no circumstances should carcasses be discarded arbitrarily.
      NOTE: Avoid room temperature exposure to prevent enzymatic degradation of biomolecules^20,21.
3. Perfusion tissue harvesting (the remaining steps are the same)
   1. Expose the heart: Lift the rat's skin tissue with your left hand and open the thoracic cavity with your right hand to fully expose the heart.
      NOTE: A deep level of anesthesia must be induced prior to initiating the thoracotomy incision for the cardiac perfusion procedure.
   2. Prepare the left ventricle for perfusion: Make a small incision on the left ventricle (size matching the perfusion needle diameter) and incise the right auricle to allow blood outflow.
   3. Perfuse saline: Insert the perfusion needle gently into the left ventricle and slowly perfuse 100 mL of saline at a flow rate of 10 mL/min until the right atrial outflow becomes clear.
   4. Perfuse fixative: Switch to 200 mL of 4% paraformaldehyde fixative, flow rate: 5 mL/min.Perfuse each rat with 100 mL of saline, followed by 200 mL of 4% paraformaldehyde. This study used a commercially prepared PFA solution. Due to the instability of PFA in solution, with significant variability potential depending on preparation and freshness, the liquid solution must be protected from light and stored at 4 °C. Use an opened aliquot promptly to ensure efficacy. If prepared from powder, the solution should be freshly made immediately before use.
      NOTE: Wear gloves, goggles, and a lab coat; perform in a fume hood because paraformaldehyde is toxic. According to the regulations of the Experimental Animal Center of Nanjing University of Chinese Medicine, paraformaldehyde waste is classified as chemical biomedical waste (Waste Code: 831-004-01). After use, it must be stored in a dedicated waste container labeled with the biohazard symbol. Centralized disposal is carried out by the university. Under no circumstances shall it be discarded improperly.
   5. Confirm perfusion success: Check for limb rigidity indicating effective fixation.
   6. Place the pancreas in a 50 mL centrifuge tube containing 30 mL of 4% paraformaldehyde solution. Fix the tissue by storing the tube stationary in the dark for 2-4 h.
   7. Transfer the pancreas to a new 50 mL centrifuge tube containing 30 mL of 70% ethanol solution. Keep the tube stationary and protected from light for approximately 20-24 h²².
   8. Paraffin embedding: Embed the tissues in paraffin wax. Store the embedded tissues at 4 °C or -20 °C.
      NOTE: This study used paraffin sections. Frozen sections are a valid alternative, but antibody performance must be verified for the specific section type beforehand to ensure optimal results.

3. Immunohistochemistry (IHC) staining of pancreas

1. Sectioning and mounting protocol
   1. Paraffin sections: Cut tissue sections at a thickness of 6 µm using a microtome and mount onto glass slides.
   2. Floatation method: Float the sections on a 40 °C tissue flotation bath to smooth wrinkles.
   3. Tissue collection: Mount sections onto adhesive-coated slides (2-3 per slide) and dry on an 80 °C slide warmer.
   4. Slide drying: Transfer slides with mounted sections to a 60 °C oven overnight after sectioning.
2. Deparaffinization and rehydration protocol
   1. Place slides in a slide rack and immerse sequentially as follows: xylene I, 7 min; xylene II, 7 min; anhydrous ethanol, 5 min; 90% ethanol, 5 min; 85% ethanol, 5 min; 75% ethanol, 5 min; distilled water, 5 min; PBS, 5 min (three times).
      NOTE: Perform xylene steps in a fume hood with gloves and goggles. Dispose of xylene waste following the identical protocol specified for paraformaldehyde as step 2.3.4.
3. Antigen retrieval protocol
   1. Preparation of sodium citrate solution: Prepare sodium citrate buffer (pH 6.0). We used a self-prepared sodium citrate buffer solution by dissolving one packet of the powder in 1 L of water.
   2. Microwave sequentially: Place the slides in a glass staining jar filled with sodium citrate buffer solution to cover the slides, then place the jar in the microwave. The samples were subjected to microwave treatment under the following sequence: medium heat (600 W) for 8 min, followed by standing without heating for 8 min, and finally low heat (300 W) for 7 min.
   3. Cool naturally to room temperature: Allow the container to cool naturally to room temperature.
   4. PBS: Rinse slides in PBS for 5 min for two times.
4. Oven incubation
   1. H₂O₂: Apply 3% H₂O₂ to the tissue sections for 10 min to block endogenous peroxidase activity. Apply approximately 50 - 80 µL of H₂O₂ to each slide.
   2. PBS: Wash slides in PBS for 5 min three times.
   3. Strept-Avidin-Biotin-Peroxidase Complex (SABC): Apply SABC dropwise onto the tissue and stain for 30 min in a 37 °C incubator. Apply approximately 50 - 80 µL of SABC to each slide.
   4. PBS: Wash slides in PBS for 5 min three times.
5. Color rendering
   1. 3,3'-diaminobenzidine (DAB): Apply DAB chromogenic solution to the tissue sections and monitor until brownish-yellow coloration develops. Stain the sections for approximately 1-5 min. Adjust the staining duration based on tissue specificity and ambient temperature. For pancreatic tissue, a staining time of 2 min at 22 °C is recommended.
   2. Rinse: Rinse slides immediately under a gentle stream of distilled water to stop the reaction.
   3. Immerse: Immerse slides in distilled water for 5 min to remove residual reagents.
6. Hematoxylin staining
   1. Hematoxylin: Immerse slides in the hematoxylin for 1 min.Hematoxylin solution is prepared using pure water at a ratio of 56 mg/mL.
   2. Rinse: Rinse slides twice in distilled water (5 s per rinse).
   3. Hydrochloric Acid-Alcohol Differentiation: Differentiate the slides in the hydrochloric acid-alcohol differentiation solution for 1 s.
   4. Immerse: Immerse the slides in tap water for 10 min to develop stable blue nuclear staining.
7. Dehydration: Dehydrate the slides sequentially through the following solutions: 70% ethanol, 80% ethanol, 90% ethanol, anhydrous ethanol, xylene I, and xylene II, for 3 minutes in each solution, respectively.
   NOTE: Perform xylene steps in a fume hood with gloves and goggles.
8. Sealing
   1. Mount the sections with neutral balsam: Apply neutral balsam to the slide using a rubber bulb dropper. Cover with a coverslip. Avoid compressing the bulb forcefully to prevent bubble formation. Apply approximately 50 µL of neutral balsam to each slide.
   2. Air-dry: Place the slides in a fume hood overnight to allow the neutral resin to dry completely.

4. Western blotting (WB) analysis

1. Decomposition of tissues
   1. Take 200 mg of rat pancreatic tissue, place it into a 2 mL grinding tube containing 1 mL of lysis buffer and 3 small steel balls. The lysis buffer is composed of complete Protease Inhibitor Cocktail and RIPA lysis buffer. Mixing ratio is 1 tablet of protease inhibitor: 50 mL RIPA lysis buffer.
   2. Grinding was performed using a ball mill with the following parameters: frequency: 30/s; time: 30 s; 10-15 times. Until no white particles are visible to the naked eye.
   3. Transfer the upper protein supernatant into a 1.5 mL micro-centrifuge tube, do not take any foam. Centrifuge it with 16900 x g 30min, 4 °C.
      NOTE: Homogenates for Western blotting analysis were prepared from the entire pancreas.
2. Protein quantification: Quantify protein concentration using the Pierce BCA Protein Assay Kit according to the manufacturer's instructions. The mixing ratio for BCA working solution is Solution A: Solution B = 50:1.
3. Protein electrophoresis:
   1. Add the prepared pancreatic protein samples (a volume of 6 µL was loaded into each lane) to sodium dodecyl sulfate-polyacrylamide gel-Young PAGE, Bis-Tirs, 10 × 8, for electrophoresis.
   2. Run at 70 V for 0.5 h, then run at 110 V for 1 h using Mini-PROTEAN Tetra System. Dilute the MOPS electrophoresis Running Buffer liquid 20×with pure water to 1× concentration.
4. Membrane transfer
   1. Dilute the transfer concentrate eBlot L2 5× to 1× working concentration using pure water. This 1× transfer buffer can be reused up to 3 times. Dilute the balance concentrate eBlot L2 5× to 1× working concentration using pure water.
   2. Cut a PVDF membrane (Immobilon-P Transfer Membrane) to the appropriate size (e.g., 7.5 cm × 5.5 cm). The pore size is 0.45 um.
   3. Pour approximately 30 mL of methanol into an incubation box with 90 × 60 × 32 mm. Immerse the PVDF membrane in the Methanol for 5 s to activate it.
   4. Pour approximately 30 mL of the prepared 1x balance solution into another clean incubation box. Carefully remove the gel from the electrophoresis plates and place it into the balance solution.
   5. Transfer the activated PVDF membrane from the methanol and also place it into the incubation box containing the balance solution. Ensure the membrane is positioned beneath the gel for proper contact. Allow both to equilibrate.
   6. Following the blotting apparatus manufacturer's instructions, place the transfer "sandwich" in this order from up to bottom: sponge, gel, PVDF membrane, sponge. Ensure no air bubbles are trapped between layers. Correctly place the assembled transfer stack into the blotting apparatus chamber. Connect the power supply and run the transfer at standard mode for 15 min.
5. Block
   1. Prepare two incubation boxes, one containing approximately 15 mL of TBST (Tris-buffered saline-Tween20), and the other with about 15 mL of 5% BSA blocking solution.
   2. Preparation of TBST solution: Dissolve one pouch of Tris Buffered Saline Powder in 2 L of pure water, then add 2 mL of Tween 20.
   3. Place the PVDF membrane into the TBST solution, ensuring the side that was in contact with the gel faces up.
   4. Wash the PVDF membrane on a shaker at 40 rpm at room temperature for 3-5 min.
   5. Transfer the PVDF membrane to the blocking buffer. Block it on a shaker at 20 rpm, at room temperature for 1 h.
6. Primary antibody incubation
   1. Wash the PVDF membrane in an incubation box containing TBST, wash the membrane three times for 5 min each on a shaker at 40 rpm at room temperature.
   2. Incubate with primary antibody overnight (14-18 h) at 4 °C (1:40000, Vinculin Polyclonal antibody) in another antibody incubation box on a shaker at 20 rpm.
   3. Ensure that the antibody is diluted with 5% BSA blocking solution, prepared according to the ratio specified in section.
7. Secondary antibody incubation
   1. Place the PVDF membrane in an incubation box containing 15 mL of TBST solution. Wash the membrane three times for 5 min each on a shaker at room temperature, agitating at 40 rpm.
   2. Prepare the secondary antibody by diluting Anti-rabbit IgG, HRP-linked Antibody at 1:2000 in 5% BSA blocking buffer.
   3. Incubate for 1 h at room temperature on a shaker at 20 rpm²³.
   4. Wash the PVDF membrane three times with TBST solution for 5 min each, following the method described above.
8. Detection
   1. Prepare the working substrate solution by mixing AccuWest ECL Plus, Raegent A and AccuWest ECL Plus, Raegent B at a 1:1 volume ratio directly in an incubation box. Perform all subsequent steps protected from light.
   2. Incubate the PVDF membrane in the solution for 5-10 s, then immediately detect immunoreactive bands using enhanced chemiluminescence (ECL) reagents-Vilber Fusion FX6 MINI. The typical exposure time ranges from 5 to 10 s.

Results

We describe a reproducible protocol for establishing a simple obesity model that reliably exhibits fatty pancreas (Figure 1). The entire process, from the animal feeding and tissue collection to the subsequent molecular validation, is schematically summarized in Figure 1. The weekly weight changes of the model group (n = 5) and the control group (n = 5) were recorded, and there was no significant difference in the weight of the two groups before feeding with HFD...

Discussion

As the primary driver of metabolic syndrome, uncomplicated obesity represents the initial manifestation of systemic lipid metabolism dysregulation. Without intervention, it may progress to T2DM and severe diabetic complications, underscoring the necessity of early metabolic correction³⁰. Pancreatic steatosis (fatty pancreas), an early marker of ectopic lipid deposition in obesity, redefines the pancreas as a non-classical adipose organ, challenging the conventional adipo-hepatic axis paradigm. Thi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
1.5 mL micro-centrifuge tube	biosharp bioengineering co. ltd	BS-15-M	WB
2ml freezing tube	LABSELECT	CV-002-200-EX	WB
2ml grinding tube	Wuhan Servicebio Technology Co., Ltd	HT-200-M	WB
3% H2O2	Booster Electronics Technology Co.,Ltd.	20A23C	IHC
4% paraformaldehyde	biosharp bioengineering co. ltd	BL539A	perfusion pancreas dissection operation
5% BSA solution	Biofroxx	4240GR100	IHC
50 mL centrifuge tube	biosharp bioengineering co. ltd	BS-500-M	tissue preservation
AccuWest ECL Plus, Raegent A	Nanjing Gene Easy Biotechnology Co., Ltd.	Q201220	WB
AccuWest ECL Plus, Raegent B	Nanjing Gene Easy Biotechnology Co., Ltd.	Q201220	WB
anhydrous ethanol	Shanghai SCR-Biotech Co., Ltd.	10009257	IHC
Anti-rabbit IgG, HRP-linked Antibody	Cell Signaling Technology.Inc,	7074	WB
cOmplete Protease Inhibitor Cocktail	Hoffmann-La Roche Ltd	82256300	WB
coverslip	CITOTEST LABWARE MANUFACTURING Co., LTD	10212450C	IHC
DAB chromogenic solution	Booster Electronics Technology Co.,Ltd.	AR2017-1	IHC
forceps	Shenzhen RWD Life Science Co., Ltd.	SP0007-M	dissection of SD rat
glass slides	CITOTEST LABWARE MANUFACTURING Co., LTD	188105	IHC
glass staining jar	Shanghai Beyotime Biotechnology Co., Ltd.	FG010	IHC
Hematoxylin	Shanghai Beyotime Biotechnology Co., Ltd.	ST2067-20g	IHC
high-fat diet	Suzhou Shuangshi Laboratory Animal Feed Technology Co., Ltd.	Table1	feeding on experimental animals
hydrochloric acid alcohol	Shanghai Beyotime Biotechnology Co., Ltd.	C0163M	IHC
isoflurane	Shenzhen RWD Life Science Co., Ltd.	R510-22-10	anesthetized rats
methanol	Biofroxx	67-56-1	WB
MOPS Running Buffer liquid 20×	Nanjing Genscript Biotechnology Co., Ltd.	C36842504	WB
neutral balsam	Beijing Solarbio Science & Technology Co., Ltd.	G8590	IHC
Neutral resin	Shanghai SCR-Biotech Co., Ltd.	G8590	IHC
PBS solution	Jiangsu Keygen Biotech Corp., Ltd	KGL2206-500	tissue preservation
Pierce BCA Protein Assay Kit	Thermo Fisher Scientific Inc, Waltham, USA.	ZB382867
Pierce BCA Protein Assay Kit	hermo Fisher Scientific Inc, Waltham, USA	ZB382867	WB
Protease Inhibitor Cocktail	Hoffmann-La Roche Ltd	82256300
PVDF membrane (Immobilon-P Transfer Membrane, Millipore, 0000380346)	Merck Millipore	0000380346	WB
RIPA lysis buffer	biosharp bioengineering co. ltd	BL504A, 70096436	WB
SABC	Booster Electronics Technology Co.,Ltd.	20C03A	IHC
saline	SHANXI SHENGAO ANIMAL PHARMACEUTICAL CO.,LTD.	270071460	tissue preservation
SD male Rats	Jiangsu GemPharmatech Co., Ltd.	D000017	rats
sodium citrate	Merck Life Science	S1804	IHC
standard maintenance feed	Suzhou Shuangshi Laboratory Animal Feed Technology Co., Ltd.	Table2	feeding on experimental animals
the balance concentrate eBlot L2 5×	Nanjing Genscript Biotechnology Co., Ltd.	C97212410	WB
the transfer concentrate eBlot L2 5×	Nanjing Genscript Biotechnology Co., Ltd.	C97211504	WB
Tris Buffered Saline, Powder	biosharp bioengineering co. ltd	BL602A	WB
Tween 20	Biofroxx	EZ681A3EA8	WB
urethane	Sigma-Aldrich Co., Ltd.	U2500-100G	anesthetized rats
Vinculin Polyclonal antibody	Proteintech Group, Inc,	26520-1-AP	WB
xylene	Nanjing Reagent Corporation	C0430530223	IHC
Young PAGE, Bis-Tirs, 10×8	Nanjing Genscript Biotechnology Co., Ltd.	C33152505	WB
Equipment
anesthesia machine	Shenzhen RWD Life Science Co., Ltd.	Model R5805	anesthetized rats
ball mill	Retsch Technology GmbH - Microtrac	MM400	WB
Centrifuges	Eppendorf SE	5418R	WB
computerized biological tissue dryer	Zhejiang Jinhua Kedi Instrument Co.	SC2013-G-KIT	IHC
enhanced chemiluminescence (ECL) reagents	VILBER BIO IMAGING	00170961	WB
microtome	Thermo Fisher Scientific Inc, Waltham, USA.	HM325	IHC
microwave	Shanghai Boxun Industry Co., Ltd.	97000	IHC
Mini-PROTEAN Tetra System	Bio-Rad Laboratories, Inc.	1658004	WB
oven	Shanghai Jinghong Scientific Co., Ltd.	DNP-9082	IHC
pure water	Sichuan UPT Ultra-Pure Technology Co., Ltd.	ULUP-TEZ	IHC