STEP 1: Euthanasia
When mice have reached their endpoint, euthanize them with a lethal injection of pentobarbital (i.p. 10 mg/kg).
STEP 2: Extraction of blood
The extraction of blood is performed via cardiac puncture. Scruff the mouse from the back with the body hanging straight vertically. Insert a 22-25G needle with a 3 mL syringe just under the ribs at the body centerline. The needle should hit the heart approximately at the level of the elbow (Figure 1). Gently, withdraw the syringe plunger until blood starts to come out. If blood doesn’t come out, move the syringe slightly upwards or downwards until blood is readily withdrawn. A volume of 1-2 mL can be extracted with this technique. Do not apply additional back pressure until the blood has filled the syringe1,2.
CRITICAL STEP: proper mouse restrain is important to ensure successful blood collection. This step needs to be done as fast as possible to ensure that vessels won’t collapse before the transcardial perfusion.
CAUTION: applying too much back pressure can collapse the heart muscle thus interfering with the blood extraction.
Rapidly transfer the blood to a vacutainer blood collection tube with EDTA. If the blood will be used for RNA extraction, apply three parts of Trizol LS per one part of blood, and process immediately or snap freeze for later processing. If the blood will sit for more than a minute in the syringe before transferring to a container with EDTA, then it may be advisable to add some EDTA to the syringe to coat the inside.
STOP POINT: samples can be stored at -80ºC until further processing or can be processed fresh. Proceed to extract RNA with a preferred method.
STEP 3: Transcardial Perfusion
Place the mouse on its back on a grate sitting on top of the rectangular bucket and tape the forelimbs and hindlimbs firmly to the grate. Perform a cut from below the ribs to the upper chest in order to expose the chest cavity. Attach hemostats to the ribs and retract in order to fully expose the heart. Insert a 21G needle attached to a perfusion pump containing 1X PBS into the left ventricle, and then perform a small cut in the right atrium. Start to pump 1X PBS to the mouse, until the fluid runs clear out of the cut in the right atrium. The liver changing from a deep red to a paler shade is an indicator of a good perfusion.
CRITICAL STEP: further perfusion with 4% PFA should be avoided as half of the brain will be used for protein extraction and immunostaining results have shown significant improvement when skipping this step.
STEP 4: Extraction of brain for immunostaining and protein/RNA
After perfusion with 1X PBS, the brain through the cranial window should look pale. Carefully remove the cranial window with forceps. Decapitate with scissors, then cut the muscles of the neck and the skin that covers the cranium. Insert the scissors through the foramen magnum, and gently start cutting the cranial bone, up to the rostral side.
CAUTION: Take care to not damage the brain during this process.
With forceps, remove the lateral bone flaps and expose the brain completely. Slide curved narrow forceps under the brain and carefully tilt it upward. When the brain is sufficiently loose, slide it out of the cranium with the help of the forceps and add to a clean petri dish sitting in an ice bath and wash the brain with ice cold 1X PBS to remove all hairs or other debris. With a clean razor blade, section the brain in half along the sagittal axis. With the help of curved forceps, carefully separate the cortex from the hippocampus (for more details see5) or dissect other brain regions are desired.
Immerse one half (for immunostaining) in a tube containing 4% PFA and fix overnight at 4°C. After 24 hours, transfer the brain to a solution containing 30% sucrose. The other half or remaining brain sections can be snap freeze in liquid nitrogen or processed immediately for RNA or protein extraction.
STOP POINT: If samples cannot be immediately processed, we recommend homogenizing the tissue immediately using a Dounce homogenizer (or other methods, such as bead mill) in the appropriate buffer, making sure to add RNAase or protease inhibitors depending on need, and then snap freeze and store at -80ºC.
STEP 5: Sample preparation
For RNA analysis, prepare a tube containing Trizol or a lysis buffer with an RNAase inhibitor of preference3.
For protein extraction, prepare extraction buffer in a 50 mL conical tube containing 1 mM AESBF (or other appropriate proteinase inhibitors), 1 tablet of cOmplete and 2 mL of RIPA buffer and dilute 1:25 with 1X PBS.
Place half brain inside a clean Dounce homogenizer and homogenize the brain with 500 uL of extraction buffer. Use small strokes to avoid bubbles. A total of 20-30 strokes should be enough to homogenize the tissue. With a pipette, transfer the homogenized tissue to an Eppendorf and keep on ice. Wash the homogenizer with MiliQ water between samples. Sonicate tubes on water for 10 minutes. Transfer samples to a 4ºC centrifuge, and centrifuge at 14000 rpm (table top centrifuge) for 30 min. Transfer the supernatant to a new tube and if desired, the pellet can be stored for further processing. If extracting protein or RNA from cerebral microvessels is of interest, follow this detailed protocol on extracting cerebral microvessels from cortex4 .
After sample preparation:
· Proceed with preferred protocol for protein measurement (Figure 2). We use Pierce BCA Protein Assay Kit (ThermoFisher CAT No. 23225)
· If performing RNA extraction, process samples as quickly as possible and clean all surfaces with RNAzap (ThermoFisher, CAT No. AM9780). Proceed with preferred protocol for RNA extraction. (Figure 3)
CRITICAL STEP: The extraction and sectioning of the brain has to be performed as quickly as possible in order to ensure the least amount of RNA or protein degradation.
STEP 5: Brain processing and slicing for immunostaining
The half brain stored in sucrose will be used for immunostaining. Extract the brain from the sucrose solution and add to a petri dish. Place the brain in a coronal or sagittal position inside the metallic cryotome holder with OCT compound as an adhesive. Let freeze inside the cryotome.
CRITICAL STEP: the brain should be oriented straight and parallel to the blade in order to ensure correct slicing.
If sections are used as serial sections prepare six tubes for a half brain, or if they will be stored as regions of interest label the tubes accordingly and fill them up with 1.7 mL of cryoprotection solution. Prepare cryoprotection solution by mixing 150 g of sucrose, 200 mL of 1X PBS, 150 mL of ethylene glycol, and complete to a final volume of 500 mL with 1X PBS. Store at 4ºC.
Position the metallic holder into the cryotome. Start trimming the OCT adhesive until the brain appears. Start slicing the brain with a thickness of 30 µm, or as desired. Select each slice, submerge in PBS and alternate the storage of each slice into different tubes. This will ensure that a representation of the whole brain is stored on each tube.
STOP POINT: Slices can be stored at 4ºC for as long as needed before mounting.
STEP 6: Immunostaining for astrocytes, microglia and amyloid plaques
Take one of the tubes stored at 4ºC and place the brain slices on a petri dish with 1X PBS. Mount the slices on a glass slide with a brush. A total of 3-6 slices can be mounted on a glass slide, depending on the size of the slices. Ensure that the slices are completely straight and not folded. Make sure to leave enough spaces in the edges for the liquid blocker marker. Allow to thoroughly dry.
After slices have dried, draw a rectangle with liquid blocking marker to prevent leakage of solutions. Wash slices 6 times with PBS for 5 minutes. Prepare a blocking solution by adding 250 µL of goat serum, 50 µL of 10% Triton X and complete to a 5 mL volume using 1X PBS. Block slides for 30 min with the blocking solution. Aspirate the blocking solution and apply primary antibody (1:500 dilution, or the appropriate dilution for the antibody, in blocking solution). Let incubate overnight at 4ºC (or as appropriate for the antibody) with a wet paper towel to avoid desiccation.
The following day aspirate the primary antibody. Wash slides three times with for 5 min with 1X PBS.
CRITICAL STEP: once the slices are thoroughly adhered after the first drying step, do not allow slides to completely dry again because this can compromise the integrity of the tissue and the quality of the staining. Make sure to cover slides completely with the solution.
Apply secondary antibody (1:500 dilution, or as appropriate, in blocking solution) and incubate for 2 hours at room temperature (or as appropriate) in a light shielded area with a wet paper towel to avoid desiccation.
Aspirate the secondary antibody and wash three times with 1X PBS. This would be an appropriate time to apply DAPI or other water-soluble counterstains. For example, if this was an Alzheimer’s disease mouse model and labeling of amyloid plaques was desired, apply Methoxy-X04 diluted 1:250 in 1X PBS and incubate at room temperature for 15 minutes. Wash 2 times with 80% ethanol. Then, allow slides to thoroughly dry before mounting. Use a mounting medium of preference (we recommend Prolong Gold). Our images were acquired with a Zeiss LSM 710 Confocal, using fluorescently-labeled secondary antibodies (Figure 4).
Katika hatua ya kwanza, euthanasia hufanywa kwa kutumia dawa ya pentobarbital. 10 mg kwa kilo. Hatua ya 2: Kuchukua damu: Kuchukua damu kwa njia ya punctuation ya moyo. Scruff panya kutoka nyuma na mwili kunyongwa moja kwa moja wima. Kuingiza sindano ya 22-25G na sindano ya 3 ml chini ya mbavu kwenye mstari wa kati wa mwili. Kinywaji cha sindano kinapaswa kugonga moyo karibu na kiwango cha kiwiko (Picha ya 1). Kwa upole, vua plunger ya sindano hadi damu ianze kutiririka. Kama damu haitoi nje, kuhamisha sindano kidogo juu au chini mpaka damu ni kwa urahisi kuondolewa. Kiasi cha 1-2 ml kinaweza kuchukuliwa kwa mbinu hii. Usisumbue damu hadi damu iwe imejaza sindano. Hatua muhimu: kuzuia panya sahihi ni muhimu ili kuhakikisha mafanikio ya mkusanyiko wa damu. Hatua hii inahitaji kufanyika haraka iwezekanavyo ili kuhakikisha kwamba mishipa haitaanguka kabla ya perfusion transcardial. Kumbuka: kutumia shinikizo kubwa kupita kiasi kunaweza kuharibu misuli ya moyo na hivyo kuingilia uchimbaji wa damu. Kuchukua damu kutoka kwa mtihani wa damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu ya damu. Ikiwa damu itatumiwa kwa ajili ya uchimbaji wa RNA, tumia sehemu tatu za Trizol-L-A na sehemu moja ya damu, na ufanye kazi mara moja au uhifadhi kwa ajili ya usindikaji wa baadaye. Ikiwa damu itakaa kwa zaidi ya dakika moja katika sindano kabla ya kuhamishwa kwenye chombo na EDTA, basi inaweza kuwa na busara kuongeza EDTA kwenye sindano ili kufunika ndani. Kwa mfano, sampuli inaweza kuhifadhiwa katika -80 ° C hadi usindikaji wa ziada au inaweza kusindika safi. Kuendelea kuchimba RNA na mbinu preferred. Hatua ya 3: Transcardial Perfusion: Weka panya juu ya grate juu ya ndoo ya mstatili na kuweka miguu ya mbele na nyuma kwa nguvu kwenye grate. Kufanya kukata kutoka chini ya mbavu kwa kifua cha juu ili kufunua shimo la kifua. Weka hemostats kwenye mbavu na kurudisha nyuma ili kufunua moyo kikamilifu. Kuingiza sindano ya 21G iliyounganishwa na pampu ya infusion iliyo na 1X-PBS katika ventricle ya kushoto na kisha kufanya upasuaji mdogo katika atrium ya kulia. Pampu ya 1x PBS ya panya ili kuondoa maji kutoka kwenye atrium ya kulia. Mabadiliko ya ini kutoka rangi nyekundu yenye kina kirefu hadi rangi nyeupe ni ishara ya umwagikaji mzuri. Hatua muhimu: infusion zaidi na 4% PFA inapaswa kuepukwa, kwani nusu ya ubongo itatumiwa kwa uchimbaji wa protini, na matokeo ya immunostaining yameonyesha uboreshaji mkubwa wakati wa kuruka hatua hii. Hatua ya 4: Uchimbaji wa ubongo kwa ajili ya immunostaining na protini (RNA) Baada ya infusion na 1X-PBS, ubongo kupitia dirisha la fuvu la kichwa inapaswa kuonekana nyeupe. Ondoa kwa uangalifu dirisha la fuvu la kichwa kwa kutumia forceps. Kupunguza kichwa kwa kisu, kisha kukata misuli ya shingo na ngozi inayofunika fuvu la kichwa. Weka msumari kupitia foramen magnum, na kwa upole uanze kukata mfupa wa fuvu la kichwa, hadi upande wa rostral. Jihadharini: Jihadharini usiharibu ubongo wakati wa mchakato huu. Kwa kutumia forceps, ondoa vipande vya mifupa ya upande na uonyeshe ubongo kabisa. Piga fimbo nyembamba iliyojipinda chini ya ubongo na uelekeze kwa uangalifu juu. Wakati ubongo ni kutosha huru, slide nje ya fuvu la kichwa kwa msaada wa forceps na kuongeza katika sahani safi ya petri kukaa katika bafu ya barafu na kuosha ubongo na barafu baridi 1x PBS kuondoa nywele zote au uchafu mwingine. Kwa kisu safi cha kunyoa, kata ubongo vipande viwili kando ya mhimili wa sagittal. Kwa kutumia forceps, kuondoa hippocampus na cortex, au kupasua sehemu nyingine za ubongo. Kuingiza nusu ya mimea katika bomba lenye PFA 4%, na kuweka usiku mmoja kwa 4 ° C. Baada ya masaa 24, ubongo hupaswa kwenye suluhisho lenye asilimia 30 ya sukari. Sehemu nyingine ya ubongo inaweza kufungia katika nitrojeni ya kioevu au kusindika mara moja kwa RNA au protini. Ikiwa sampuli haziwezi kusindika mara moja, tunapendekeza homogenize tishu mara moja kwa kutumia homogenizer ya Dounce (au mbinu nyingine, kama vile mill ya bead) katika buffer inayofaa, kuhakikisha kuongeza RNA au protease inhibitors kulingana na mahitaji, kisha funga na kuhifadhi kwenye -80oC. Hatua ya 5: Kuandaa sampuli kwa ajili ya uchambuzi wa RNA: Kuandaa tube yenye Trizol au buffer ya lysis na kizuizi cha RNA. Kwa ajili ya extraction ya protini, kuandaa buffer extraction katika 50 ml mole tube, ambayo ina 1 ml AESB-F (au nyingine sahihi proteinase inhibitors), 1 mguu wa cOMPLETE na 2 ml ya buffer RIPPA, na dilute 1: 25 na 1 ml ya PBS. Kuingiza nusu ya ubongo ndani ya homogenizer safi na homogenize ubongo na 500 ml ya buffer ya uchimbaji. Tumia madoa madogo ili kuepuka vifusi. Kwa jumla, vipindi 20 hadi 30 vya kupigania vinapaswa kuwa vya kutosha kuunganisha tishu. Kwa pipette, kuhamisha tishu homogenized kwa Eppendorf na kuweka juu ya barafu. Kuosha homogenizer na maji MiliQ kati ya sampuli. Sonate tubes juu ya maji kwa dakika 10. Kwa mfano, kwa centrifuge ya 4 ° C, fimbo ya mviringo ya 14000 rpm (mviringo wa mviringo wa mviringo wa mviringo) inaweza kuhifadhiwa kwa muda wa dakika 30. Kuhamisha supernatant kwa tube mpya na kama unataka, pellet inaweza kuhifadhiwa kwa ajili ya usindikaji zaidi. Kwa hivyo, ikiwa utapata protini kutoka kwa micro-vessels ya ubongo, fanya kama ilivyoelezwa hapa chini. Baada ya kutayarisha sampuli, endelea na itifaki inayopendekezwa kwa kipimo cha protini (Sura 2). Pia, mtihani wa protini ya Pierce BCA (Thermofisher CAT No. "Kama unafanya ""DNA extraction,"" chukua sampuli haraka iwezekanavyo na kusafisha nyuso zote kwa RNAZAP (ThermoFisher, CAT No. (AMR 9780) Endelea na itifaki inayopendekezwa kwa ajili ya uchimbaji wa RNA. Hatua muhimu: Uchimbaji na kugawanya ubongo lazima kufanywa haraka iwezekanavyo ili kuhakikisha kiwango cha chini cha RNA au uharibifu wa protini. Hatua ya 5: Utaratibu wa ubongo na kukata kwa ajili ya immunostaining: nusu ya ubongo iliyohifadhiwa katika sucrose itatumika kwa ajili ya immunostaining. Toa ubongo kutoka kwa suluhisho la sukari na uongeze kwenye bakuli la petri. Kuingiza ubongo katika nafasi ya sagittal au coronal ndani ya cryotome ya chuma na mchanganyiko wa OCT kama adhesive. Acha kufungia ndani ya cryotome. Hatua muhimu: ubongo unapaswa kuwa moja kwa moja na sambamba na kisu ili kuhakikisha slicing sahihi. Ikiwa sehemu zinatumiwa kama sehemu za mfululizo, kuandaa mirija sita kwa nusu ya ubongo, au ikiwa zitahifadhiwa kama maeneo ya maslahi, weka alama ya mirija hiyo na uijaze na 1.7 ml ya suluhisho la cryoprotection. Kuandaa cryoprotection ufumbuzi kwa kuchanganya 150 g ya sucrose, 200 ml ya 1XPBS, 150 ml ya ethylene glycol, na kukamilisha kwa kiasi cha mwisho cha 500 ml ya 1XPBS. Hifadhi katika 4oC. Kuweka mmiliki chuma katika cryotome. Kuondoa OCT adhesive na kuondoa nywele. Kuchukua kipande cha ubongo kwa unene wa 30 μm au kama inavyotakiwa. Chagua kila kipande, kuzamisha katika PBS na kubadilisha kuhifadhi kila kipande katika mirija tofauti. Hii itahakikisha kwamba uwakilishi wa ubongo mzima umehifadhiwa kwenye kila bomba. Kumbuka: vipande vya vipande vinaweza kuhifadhiwa kwa 4 ° C kwa muda mrefu kama inahitajika kabla ya kuunganisha. Mfano: Mfano wa astrocyte, microglia na plaques ya amyloid, na kipande cha ubongo kwenye sahani ya petri. Weka vipande hivyo kwenye kioo kwa kutumia brashi. Kwa jumla, vipande 3 hadi 6 vinaweza kuwekwa kwenye slide ya kioo, kulingana na ukubwa wa vipande. Hakikisha kwamba vipande hivyo viko sawa kabisa na si vilivyojipinda. Hakikisha kuacha nafasi ya kutosha katika kingo kwa ajili ya marker blockers kioevu. Acha kukauka kabisa. Baada ya vipande vimeuka, kuchora mstatili na marker kuzuia kioevu kuzuia uvujaji wa ufumbuzi. Fua vipande vya vipande mara sita kwa PBS kwa dakika tano. Kuongeza 250 ml ya seramu ya mbuzi, 50 ml ya Triton X ya 10% na kukamilisha kwa kiasi cha 5 ml kwa kutumia 1 ml ya PBS. Kuhifadhi slides kwa muda wa dakika 30 na ufumbuzi wa kuzuia. Kuchukua suluhisho la kuzuia na kutumia antibody ya msingi (kuchanganywa kwa 1: 500 au mchanganyiko unaofaa wa antibody katika suluhisho la kuzuia). Acha incubate usiku kwa 4 ° C (au kama inafaa kwa ajili ya kingamwili) na kitambaa cha karatasi mvua ili kuepuka desiccation. Siku iliyofuata, chukua chembe ya kwanza ya kinga. Kuosha slides mara tatu kwa muda wa dakika 5 na 1X PBS. Baada ya hatua ya kwanza ya kukausha, slides haziruhusiwi kukauka kabisa tena, kwa sababu hii inaweza kuhatarisha uadilifu wa tishu na ubora wa rangi. Hakikisha kufunika slides kabisa na ufumbuzi. Kuongeza antibody ya pili (kwa kiasi cha 1: 500 au kama inavyotakiwa, katika suluhisho la kuzuia) na incubate kwa masaa mawili katika joto la chumba (au kama inavyotakiwa) katika eneo la taa-kuhifadhiwa na kitambaa cha karatasi cha mvua ili kuepuka kukauka. Aspirate antibody ya pili na kuosha mara tatu na 1X PBS. Wakati huu ni wakati mzuri wa kutumia DAP au counter stains nyingine zinazotoweka katika maji. Kwa mfano, ikiwa hii ilikuwa mfano wa panya wa ugonjwa wa Alzheimer na kuashiria plaques ya amyloid ilitakiwa, tumia Methoxy-X4 diluted 1: 250 katika 1X PBS na incubate katika joto la chumba kwa dakika 15. Kwa kawaida, kunaweza kuosha kwa kutumia ethanol ya 80% kwa muda mrefu. Kisha, acha slaidi ziweke kavu kabisa kabla ya kuunganishwa. Tumia vyombo vya kuunganisha ya upendeleo (tunakumbusha Prolong Gold). Picha za ZEISS LSM-710 Confocal zilipigwa kwa kutumia antibodies ya pili iliyochorwa kwa fluorescent.