Evans Blue Stain (2%)伊文思藍染色液（2%）

産品信息

産品描述

伊文思藍（Evans Blue，CAS NO：314-13-6），也稱作埃文斯藍，是一(yī)種非膜滲透性的染料。當質膜受損，染料能(néng)進入細胞質和細胞核，從(cóng)而将其染成藍色，可用來檢測細胞活力。伊文思藍還(hái)能(néng)用來研究血腦(nǎo)屏障（BBB）滲透性，通(tōng)過與白(bái)蛋白(bái)結合來指示血腦(nǎo)屏障穿透蛋白(bái)能(néng)力。正常情況下(xià)，血漿白(bái)蛋白(bái)不能(néng)透過血腦(nǎo)屏障，所以染色後具完整血腦(nǎo)屏障的神經系統不著(zhe)色，而完整性被破壞的神經系統會(huì)著(zhe)色。

本品為(wèi)2%的伊文思藍染色液，根據具體用途直接使用或簡單稀釋後再使用。

保存與運輸方法

保存：2-8℃避光(guāng)保存，1年(nián)有效。 

運輸：室溫運輸。

使用方法

根據具體實驗用途，直接用伊文思藍染色液（2%）或将其用PBS稀釋到(dào)伊文思藍染色液（0.5%）再使用。

以下(xià)染色步驟以伊文思藍染色液（0.5%）為(wèi)例，僅做參考。

一(yī)、血腦(nǎo)屏障通(tōng)透性

一(yī).1 取處理後的動物(wù)（以小(xiǎo)鼠為(wèi)例），經尾靜(jìng)脈或股靜(jìng)脈按照(zhào)2~3ml/kg體重的比例注射伊文思藍染色液（0.5%） 數s至1min内，小(xiǎo)鼠眼睛、皮膚出現藍色。0.5～1h後處死小(xiǎo)鼠，取出目的腦(nǎo)組織。【注意】：伊文思藍染色液的注射用量需要根據動物(wù)類型以及體重來決定。

一(yī).2 将腦(nǎo)組織置于1.5ml離心管中，加入1ml PBS，迅速用組織勻漿器(qì)将腦(nǎo)組織制成勻漿，4℃ 1000g離心15min。

一(yī).3 取上(shàng)清，加入等量三氯乙酸，4℃孵育18~24h。該步驟亦可采用如下(xià)操作：取上(shàng)清，按上(shàng)清：丙酮=3:7比例加入丙酮，室溫孵育24h。

一(yī).4 4℃ 1000g離心20~30min（或2000g離心15min）。

一(yī).5 取上(shàng)述溶液1~2ml，用分光(guāng)光(guāng)度計測620 nm下(xià)的OD值。同時測定已知不同梯度的标準伊文思藍的OD值，繪制标準曲線。根據标準曲線計算(suàn)出待測樣品的伊文思藍含量。

二、細胞活力鑒定

2.1取100μl重懸細胞到(dào)常規離心管内，加入100μl伊文思藍染色液（0.5%）輕輕混勻，染色3min(染色時間可适當延長(cháng)，但不宜超過10min)。

2.2吸取少量經過染色後的細胞，用血細胞計數闆計數。通(tōng)常如果要比較精确地進行定量，每個(gè)細胞樣品至少數500個(gè)細胞，數出藍色細胞和細胞總數。

三、種子染色

3.1用刀片做橫切和沿種胚中央準确縱切，入染色液染色3~5min。

3.2蒸餾水(shuǐ)中浸泡20~60min，視脫色程度而定。

注意事(shì)項

1） 伊文思藍對人體有輕微毒性，操作過程中請注意防護。

2） 細胞染色時，需注意凋亡小(xiǎo)體偶爾也有拒染現象。

3） 建議用低(dī)溫冷凍離心機(jī)進行離心。

4） 為(wèi)了您的安全和健康，請穿實驗服并戴一(yī)次性手套操作。

相(xiàng)關産品

文獻示例（An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse，Ref：PMID: 30272649）

This method uses FVBN adult mice, aged 16 - 20 weeks, found to be optimal for the purposes of this study. Day 1 includes steps 1 - 5 and Day 2 includes steps 6 - 7 (Figure 1).

1. Equipment Preparation

1. Secure an adequate supply of sterile, disposable syringes and needles, if ketamine/xylazine is used as the anesthetic (as recommended). If isoflurane is used as the anesthetic, check the oxygen tank and the fluid level of isoflurane to make sure there are adequate supplies for the experiment before starting. Also, assemble the nosecone breathing circuits and attach them to the induction box; attach new charcoal canisters to the breathing circuits. Prepare the induction box by turning on the oxygen and ascertaining that the second stage reads approximately 50 psi.

2. Turn on the heating pad to 37 °C.

3. Prepare the rectal temperature probe for the surgery.

2. Mouse Preparation

This step includes anesthesia, hair removal, and positioning (adult FVBN mice-age 16 - 20 weeks).

1. Weigh the mice and record the weights.

2. Anesthetize the mice.

1. Administer ketamine and xylazine IP (80 - 100 mg/kg and 7.5 - 16 mg/kg, respectively). However, it is recommended to begin with lower doses of ketamine and xylazine (about 30 mg/kg and 6 mg/kg, respectively).

2. Maintain the anesthesia with about 0.1 - 0.25 times initial doses of ketamine/xylazine throughout the surgery. Ketamine and xylazine were the anesthetic agent(s) of choice in this particular study, as more reproducibility and survivability were observed. The anesthetic agent(s) of choice in other studies may be found to be different.  If isoflurane is used, put the mouse in an induction chamber and turn on isoflurane to 5% until the mouse loses full consciousness. Then use a nasal nosecone set at 1.5 - 2.5% isoflurane throughout the remainder of the procedure.

3. Monitor the mice every 2 - 3 min by toe pinch to check for appropriate depth of anesthesia.

4. Shave the ventral neck area of the mouse.

5. Place the mouse in a supine position on the preheated pad. Secure the paws and feet of the mouse to the surgical surface with tape.

6. Place artificial tear ointment onto the eyes to prevent drying out during surgery.

3. Surgical Details

1. Make a 1 cm incision in the right ventral neck over the jugular vein.

2. Apply one or two drops of lidocaine (1 - 2%) into the incision area for pain management and to promote vasodilation. Wait 2 min. for the lidocaine to take effect. 

3. Expose and isolate the right internal jugular vein via blunt dissection. Tie the vein off with 4-0 suture and gently retract the rostral end of the vessel with a hemostat. Cut a hole, using fine scissors, about 3 mm below or caudal to the tie, approximately halfway through the diameter of the vein.

4. Mark a PV-1 polyvinyl catheter 1.5 cm from the end and insert it into the jugular vein through the hole using a vessel dilator, and thread the catheter towards the caudal end of the vessel, approximately 1.5 cm.

5. Tie the catheter securely within the caudal part of the vessel (below the cut), with 4-0 silk. Tie the rostral part of the vessel to the outside of the catheter with the loose ends of the suture which was used to tie off the rostral jugular vein, in step 3.3.

6. Tack the skin loosely back together around the catheter with 4-0 silk to help prevent loss of body heat and desiccation of tissues.

7. Connect the catheter to a syringe containing heparinized saline (10 U/mL) and flush the catheter.

4. Injections

1. Inject the Evans blue solution (50 μL of a 30 mg/mL solution in 0.9% normal, unheparinized saline, or approximately 50 mg/kg) into the jugular vein catheter, followed by a small volume of heparinized saline to flush the line.

2. 2 min later, inject substance P (100 μL of a 0.3 μM solution in 0.9% normal, unheparinized saline, or 1 nmol/kg), followed by a small volume of heparinized saline to flush the line. Substance P augments the extravasation of plasma proteins through the endothelial layer; in this protocol, it routinely induces an augmentation of plasma extravasation values of approximately 1.5-fold, making plasma extravasation values easier to measure.

3. Wait 18 min after the substance P is injected. During this time, the Evans blue dye will equilibrate and circulate.

4. Terminate the experiment 18 min after the injection of substance P (20 min after Evans blue injection) by sacrificing the mouse with cervical dislocation. It is likely that the mouse can be directly cervically dislocated, without first giving an overdose of anesthetic, as the mouse is probably still well-anesthetized from the surgery. However, if it is necessary, an overdose of the anesthetics ketamine/xylazine, isoflurane, or pentobarbital may be given, followed by cervical dislocation.

5. Isolation of Organs

1. Cut open the chest cavity of the mouse and gravity-perfuse (from a height of about 51 cm or 20") the heart and blood vessels with 50 mL of 50 mM sodium citrate, pH 3.5. pH 3.5 presumably preserves Evans blue binding to albumin.

2. Excise 1 - 5 relevant organs (tissues) with a dissecting scalpel (e.g. urinary bladder, kidney, stomach, liver, pancreas, proximal or distal colon, ileum, duodenum, flank skin, ears, tail, heart, and/or lungs) and remove any residual contents, if present.

3. Rinse the organs in room temperature (RT) phosphate-buffered saline (PBS; 1.44 g of Na2HPO4, 0.24 g of KH2PO4, 8.0 g of NaCl, and 0.20 g of KCl in 1 L, pH 7.4).

4. Blot the organs with tissue, cut each organ in half, and weigh each half (wet weights, in g).

5. Dry one half of the tissue in a drying oven at 150 °C, on foil, for 48 h.

6. Place the other tissue half in a consistent volume (up to 200 µL) of formamide in a microfuge tube for 48 h (and up to 72 h) to extract the Evans blue.

6. Measurement of Tissue OD

1. Remove 50 µL of Evans blue-infused formamide (after 48 - 72 h RT incubation) from the microfuge tube and place into one well of a 96-well polystyrene plate. Be careful not to transfer tissue pieces along with the formamide.

2. Place 50 µL of new, pure formamide into each of two empty wells of the 96-well plate for the blanks.

3. Measure and record the OD620 of each well of the 96-well plate on an absorbance plate reader. 620 nm is the absorbance max of Evans blue.

7. Calculation of Plasma Extravasation

1. Weigh the dry tissue half which has been in the oven for 48 h.

2. Calculate the wet weight/dry weight ratio for the specific organ of interest from each individual mouse, starting with the wet weight of this tissue half (obtained in step 5.4), divided by the dry weight of this same tissue half (obtained in step 7.1).

3. Calculate the dry weight (in g) of the tissue half in formamide by dividing the wet weight of the tissue half before it was placed in formamide (obtained in step 5.4) by the wet: dry weight ratio for the specific organ of interest (calculated in step 7.2).

4. Calculate the corrected OD620 values. Starting with the OD620 value from each experimental well of the 96-well plate (containing Evans blue-infused formamide from each tissue, obtained in step 6.3), subtract the blank well OD620 value (the mean OD620 value of the two wells containing pure formamide, prepared in step 6.2, OD620 values obtained in step 6.3) from each experimental value.

5. Calculate the plasma extravasation value by dividing the corrected OD620 (calculated in step 7.4) by the dry weight of the tissue half in formamide (calculated in step 7.3). The units of plasma extravasation will be OD620/g dry weight.

6. Analyze data and express as the mean ± SEM. Statistically compare groups by t test or one-way analysis of variance and Scheffé's multiple-comparison test (lycofs01.lycoming.edu), as appropriate.

— —Written/Edited by V. Shallan【版權歸MKBio懋康所有】

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